Cell based assay for determining antibody or ligand binding and function

ABSTRACT

The present invention relates to a new cell based assay for combined determination of antibody or ligand binding and function in the same vial.

SEQUENCE LISTING

This application contains a Sequence Listing submitted via EFS-Web andhereby incorporated by reference in its entirety. Said ASCII copy,created on Jun. 25, 2018, is named P32944-US-SequenceListing.txt, and is2,227 bytes in size.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 application of International Application No.PCT/EP2016/064238, filed on Jun. 21, 2016, which claims priority toEuropean Application No. 15173929.9 filed on Jun. 25, 2015, the contentsof which are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to a new cell based assay for determiningantibody or ligand binding and function.

BACKGROUND

Chemotherapy is until now still one of the most commonly used treatmentsfor cancer. However, as chemotherapeutic reagents target activelydividing cells, which is one characteristic of cancerous cells, alsohealthy dividing cells such as blood cells, cells in the intestine,mouth and hair are affected resulting in rather strong side effects.Scientists are continuously working on the improvement of theadministration and combination of chemotherapeutic reagents to minimizethose side effects. Additionally, antibody based therapies have evolvedover the last 15 years and represent now a valuable combination oralternative to chemotherapeutic approaches in the treatment ofhematological malignancies and solid tumors. Unlike chemotherapy,antibody therapies target specific antigens on cancer cells thusallowing a more side directed treatment thereby reducing the sideeffects on healthy tissue. In the process of developing anantibody-based therapeutic reagent, various assays are required toidentify the best candidates to bring into clinical trials andeventually to the market. In a first early preclinical phase, theantibodies have to be generated and analyzed for theirtarget-specificity, as well as their affinity to the target andfunctionality. Binding properties can be analyzed using variousprotein-protein interaction assays, such as FRET-based methods, SurfacePlasmon Resonance (SPR), fluorescence-activated cell sorting (FACS) orAlpha Screen™. Functionality is generally tested in various cell-basedassays designed to mimic the physiological situation as close aspossible to identify the best candidates to be tested in animal modelsbefore entering clinical trials. These functional assays are commonlycarried out using primary cells, tumor cell lines or reporter cells thatare designed to express a reporter upon activation of a specificpathway.

Designing combination assays which allow assessment of binding andfunctionality at an early state in the development process of anantibody therapeutic molecule would be of great benefit.

The inventors of the present invention developed a novel assay whichcombines the assessment of binding and functionality of antibodies andantibody like constructs (e.g. ligands) in the same vial. This novelassay is useful for example for screening or characterization purposes.

This new assay represents a valuable tool for early screening for notonly binding but also functionality which will allow identifying thebest binders at an early stage in the development of the drug candidate.

SUMMARY

In one embodiment there is provided an in vitro assay for determiningthe binding and functionality of an antibody or a ligand specificallybinding to a target antigen comprising the following steps

i) providing cells which

-   -   a) express the target antigen on their surface,    -   b) are covalently or noncovalently labelled with an energy donor        compound and    -   c) comprise a reporter gene under the control of a response        element of the target antigen

ii) adding the antibody or ligand to be tested

iii) measuring the binding to the target antigen by determining theenergy transfer, wherein the energy acceptor compound is covalently ornoncovalently conjugated either to the antibody to be tested or to asecondary antibody binding to the first antibody; and

iv) determining functionality of the antibody or ligand by correlatingthe level of the expression of the reporter gene with the level oftarget antigen activation or inhibition.

In one embodiment the energy donor and acceptor compound are afluorescent resonance energy transfer (FRET) energy donor and acceptorcompound and the energy transfer determined in step iii) is fluorescentresonance energy transfer (FRET). In one embodiment the FRET is timeresolved FRET.

In one embodiment the FRET energy donor compound is Terbium cryptateand/or the FRET energy acceptor compound is d2.

In one embodiment the energy donor and acceptor compound are abioluminescence energy transfer (BRET) energy donor and acceptorcompound and the energy transfer determined in step iii) isbioluminescence energy transfer (BRET).

In one embodiment the energy donor and acceptor compound are an alphascreen acceptor and donor bead and the energy transfer determined instep iii) is an energy transfer from a singlet oxygen to an thioxenederivative within the acceptor bead.

In one embodiment the binding to the target antigen and thefunctionality of the antibody are measured in the same vial.

In one embodiment the target antigen is covalently or noncovalentlylabelled with the energy donor compound.

In one embodiment the energy donor compound is covalently ornoncovalently linked to wheat germ agglutinin (WGA).

In one embodiment the reporter gene is selected from a gene coding for afluorescent protein or a gene coding for an enzyme whose catalyticactivity can be detected.

In one embodiment the reporter gene is coding for green fluorescentprotein (GFP) or luciferase.

In one embodiment the target antigen is a cell surface receptor.

In one embodiment steps iii) and iv) are performed consecutively orsimultaneously.

In one embodiment the target antigen and the response element are partof the NF-κB pathway.

In one embodiment the response element comprises at least one DNA repeatwith a DNA sequence of SEQ ID NO: 1, 2, 3, 4 or 5.

In one embodiment the response element comprises a DNA sequence of SEQID NO 6, 7, 8 or 9.

In one embodiment the assay comprises the preliminary step oftransfection of the cells with an expression vector comprising the DNAsequence coding for the reporter gene under the control of the targetantigen response element.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Determination of the transfection efficiency as well asviability measurement of the cells after SNAP-Receptor X transfectionand labeling with terbium, a donor fluorescent dye. The terbium signalwas measured for 10 000 cells per well at a wavelength of 615 nm. Thetransfection reagents Lipofectamine 2000, Lipofectamine LTX and XtremeGene HP were compared.

FIG. 2 . Determination of the transfection efficiency of LipofectamineLTX as well as viability measurement of HEK NFκB-Luc-GFP and HeLaNFκB-Luc cells after SNAP-Receptor X transfection and labeling withterbium, a donor fluorescent dye. The terbium signal was measured with10 000 cells per well at a wavelength of 615 nm.

FIG. 3 . Tag-lite® indirect binding assay with Antibody-I andAntibody-II to Receptor X expressing HEK NFκB-Luc-GFP and HeLa NFκB-Luccells. The antibodies were diluted ranging from 25 nM to 0.05 nM in atwo-fold dilution series. A concentration of 150 nM for the d2-labeledsecondary antibody was used. The binding curve was fitted by nonlinearregression with Graph Pad Prism 6.0.

FIG. 4 . Indirect Tag-lite® binding assay with Antibody-I binding toReceptor X expressing HEK NFκB-Luc-GFP cells. Antibody-I was dilutedfrom 12.5 nM to 0.025 nM in a two-fold dilution series. The d2-labeledsecondary antibody was used in a concentration of 150 nM or 75 nM. Thebinding curve was fitted by nonlinear regression with Graph Pad Prism6.0.

FIG. 5 . Indirect Tag-lite® binding assay with Antibody-I, Antibody-IIIand Antibody-IV to Receptor X expressing HEK NFκB-Luc-GFP cells. Allantibodies were diluted from 20 nM to 0.04 nM in a two-fold dilutionseries. The d2-labeled secondary antibody was used at a concentration of75 nM. The binding curve was fitted by nonlinear regression with GraphPad Prism 6.0.

FIG. 6 . Indirect Tag-lite® binding assay of Antibody-III to Receptor Xexpressing HEK NFκB-Luc-GFP cells. The antibody was diluted at aconcentration ranging from 20 nM to 0.04 nM in a two-fold dilutionseries. The d2-labeled secondary antibody was used in a concentration of75 nM. Incubation at room temperature was compared to incubation at 37°C. The binding curve was fitted by nonlinear regression with Graph PadPrism 6.0.

FIG. 7 . Indirect Tag-lite® binding assay of Antibody-III binding toReceptor X expressing HEK NFκB-Luc-GFP cells. The antibody was dilutedat a concentration ranging from 20 nM to 0.04 nM in a two-fold dilutionseries. The d2-labeled secondary antibody was used at a concentration of75 nM. Different buffers and media for the dilutions were compared. Thebinding curve was fitted by nonlinear regression in Graph Pad Prism 6.0.

FIG. 8 . Indirect Tag-lite® binding assay of Antibody-III binding toReceptor X expressing HEK NFκB-Luc-GFP cells. The experiment wasperformed directly after thawing the cells and every following day for3. The antibody was diluted ranging from 20 nM to 0.04 nM in a two-folddilution series. The d2-labeled secondary antibody was used at aconcentration of 75 nM. The binding curve was fitted by nonlinearregression with Graph Pad Prism 6.0.

FIG. 9 . Determination of the terbium signal and viability of labeledReceptor X expressing HEK NFκB-Luc-GFP cells. The terbium signal wasmeasured directly after thawing and each following day until day 3. 10000 cells per well were seeded every day and measured at a wavelength of615 nm.

FIG. 10 . Luciferase 1000 assay system using Antibody-I and TNFα toReceptor X expressing HEK NFκB-Luc-GFP cells after 6 h of incubation.The dilution series of TNFα (two-fold) ranged from 25 to 0.8 ng/ml, theone of Antibody-I (four-fold) ranged from 30 to 0.03 nM. Antibody-I andthe secondary antibody alone were used as controls. The binding curvewas fitted by nonlinear regression with Graph Pad Prism 6.0.

FIG. 11 . ONE-Glo™ Luciferase assay system compared to Luciferase 1000assay system after 48 h of incubation upon activation by TNF-α. The NFκBpathway of stably transfected HEK NFκB-Luc-GFP-Receptor X cells andtransiently transfected HEK NFκB-Luc-GFP-Receptor X-Tb cells wasactivated through TNFα (50 ng/ml).

FIG. 12 . ONE-Glo™ Luciferase assay system after 6 h and 48 h ofincubation. The NFκB pathway of stably transfected HEKNFκB-Luc-GFP-Receptor X cells and transiently transfected HEKNFκB-Luc-GFP-Receptor X-Tb cells was activated by Antibody-III (10 nM)as a primary antibody and anti-hu IgG Fcγ-specific goat IgG F(ab)2 as asecondary antibody (40 nM).

FIG. 13 . ONE-Glo™ Luciferase assay system performed in a 96-well-plateas well as in a 384-well-plate after 24 h of incubation. The NFκBpathway of stably transfected HEK NFκB-Luc-GFP-Receptor X cells wasactivated through Antibody-III (10 nM) as a primary antibody and anti-huIgG Fcγ-specific goat IgG F(ab)2 as a secondary antibody (40 nM).

FIG. 14 . ONE-Glo™ Luciferase assay system performed in a 384-well-plateafter 6 h of incubation. The NFκB pathway of stably transfected HEKNFκB-Luc-GFP-Receptor X cells was activated through Antibody-III (10 nM)as a primary antibody and anti-hu IgG Fcγ-specific goat IgG F(ab)2 as asecondary antibody (40 nM).

FIG. 15 . WGA-HTRF indirect binding assay with Antibody-III to ReceptorX expressing HEK NFκB-Luc-GFP cells. Antibody-V served as a negativecontrol. Both antibodies were diluted ranging from 1.56 nM to 0.01 nM ina two-fold dilution series. The d2-labeled secondary antibody was usedin a concentration of 75 nM. The binding curve was fitted by nonlinearregression with Graph Pad Prism 6.0.

FIG. 16 . Indirect Tag-lite® binding assay with Antibody-III fortransiently supertransfected and transiently transfected Receptor Xexpressing HEK NFκB-Luc-GFP cells. All antibodies were diluted rangingfrom 12.5 nM to 0.02 nM in a two-fold dilution series. The d2-labeledsecondary antibody was used in a concentration of 75 nM. The bindingcurve was fitted by nonlinear regression with Graph Pad Prism 6.0.

FIG. 17 . Stably transfected, transiently transfected and transientlysupertransfected Receptor X expressing HEK NFκB-Luc-GFP cells weretested in a ONE-Glo™ luciferase assay system after 24 h of incubation.The NFκB pathway was activated through Antibody-III in a two-folddilution series ranging from 40 to 2.5 nM. Anti-hu IgG Fcγ-specific goatIgG F(ab)2 as a secondary antibody was added in a four-fold molar extentcompared to the primary antibody.

FIG. 18 . Transiently supertransfected Receptor X expressing HEKNFκB-Luc-GFP cells were tested in an ONE-Glo™ Luciferase assay systemafter 24 h of incubation. The NFκB pathway was activated throughAntibody-III in a concentration of 40 nM. Anti-hu IgG Fcγ-specific goatIgG F(ab)2 and anti-hu-IgG-d2 as secondary antibodies were tested inratios between 1:1 and 1:5 (1°: 2° antibody).

FIG. 19 . Indirect Tag-lite® binding assay with Antibody-III totransiently supertransfected Receptor X expressing HEK NFκB-Luc-GFPcells. Antibody-III was diluted ranging from 40 nM to 0.02 nM in atwo-fold dilution series. The d2-labeled secondary antibody was used ina concentration of 120 nM. The binding curve was fitted by nonlinearregression with Graph Pad Prism 6.0.

FIG. 20 . Transiently supertransfected Receptor X expressing HEKNFκB-Luc-GFP cells were tested in a ONE-Glo™ luciferase assay systemafter 24 h of incubation. The NFκB pathway was activated throughAntibody-III in a two-fold dilution series ranging from 40 to 0.02 nM.Anti-human-IgG-d2 as a secondary antibody was added in a three-foldmolar extent compared to the primary antibody.

FIG. 21 . AlphaScreen binding assay with Drozitumab and control antibodyAbY to huDR5 expressing HEK EBNA cells. All antibodies were dilutedstarting from 0.63 nM to 0.05 nM final in well in 1 in 2 dilutions. (A)Sigmoidal binding curve of Drozitumab and AbY fitted by nonlinearregression. (B) Determination of the KD values of Drozitumab and AbY bynonlinear regression.

FIG. 22 . AlphaScreen binding assay with AbZ and control antibody AbY toreceptor Z expressing HEK EBNA cells. All antibodies were dilutedstarting from 2.5 nM to 0.05 nM final in well in 1 in 2 dilutions. (A)Sigmoidal binding curve of AbZ and AbY fitted by nonlinear regression.(B) Determination of the KD values of AbZ and AbY by nonlinearregression.

FIG. 23 . WGA-FRET binding assay of anti-CD3 to CD3 expressing cells ineither PBS/1% FCS (a) or growth medium (b). Additionally, a non-bindingcontrol IgG was included in both assays as negative control. The curveswere fitted with Graph Pad Prism one site specific binding function.

FIG. 24 . Activation of the NFkB pathway upon binding of anti-CD3 eitherin PBS/1% FCS (a) or growth medium (b). Additionally, a non bindingcontrol IgG was used as negative control in both assays. The Luciferaseactivity was measured using the ONE-Glo™ Luciferase assay system. TheEC50 was determined using GraphPad Prism sigmoidal dose response fit.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

As used herein, a “reporter gene” means a gene whose expression can beassayed. In one preferred embodiment a “reporter gene” is a gene thatencodes a protein the production and detection of which is used as asurrogate to detect indirectly the activity of the antibody or ligand tobe tested. The reporter protein is that protein encoded by the reportergene. Preferably, the reporter gene encodes an enzyme whose catalyticactivity can be detected by a simple assay method or a protein with aproperty such as intrinsic fluorescence so that expression of thereporter gene can be detected in a simple and rapid assay requiringminimal sample preparation. Non-limiting examples of enzymes whosecatalytic activity can be detected are Luciferase, beta Galactosidase,Alkaline Phosphatase. Luciferase is a monomeric enzyme with a molecularweight (MW) of 61 kDa. It acts as a catalysator and is able to convertD-luciferin in the presence of Adenosine triphosphate (ATP) and Mg2+ toluciferyl adenylate. In addition, pyrophosphate (PPi) and adenosinemonophosphate (AMP) are generated as byproducts. The intermediateluciferyl adenylate is then oxidized to oxyluciferin, carbon dioxide(CO2) and light. Oxyluciferin is a bioluminescent product which can bequantitatively measured in a luminometer by the light released from thereaction. Luciferase reporter assays are commercially available andknown in the art, e.g. Luciferase 1000 Assay System and ONE-Glo™Luciferase Assay System.

The term “protein with intrinsic fluorescence” refers to a proteincapable of forming a highly fluorescent, intrinsic chromophore eitherthrough the cyclization and oxidation of internal amino acids within theprotein or via the enzymatic addition of a fluorescent co-factor. Theterm “protein with intrinsic fluorescence” includes wild-typefluorescent proteins and mutants that exhibit altered spectral orphysical properties. The term does not include proteins that exhibitweak fluorescence by virtue only of the fluorescence contribution ofnon-modified tyrosine, tryptophan, histidine and phenylalanine groupswithin the protein. Proteins with intrinsic fluorescence are known inthe art, e.g. green fluorescent protein (GFP), red fluorescent protein(RFP), Blue fluorescent protein (BFP, Heim et al. 1994, 1996), a cyanfluorescent variant known as CFP (Heim et al. 1996; Tsien 1998); ayellow fluorescent variant known as YFP (Oruro et al. 1996; Wachter etal. 1998); a violet-excitable green fluorescent variant known asSapphire (Tsien 1998; Zapata-Hommer et al. 2003); and a cyan-excitablegreen fluorescing variant known as enhanced green fluorescent protein orEGFP (Yang et al. 1996) and can be measured e.g. by live cell imaging(e.g. Incucyte) or fluorescent spectrophotometry.

As used herein, the term “functionality of an antibody or ligand” refersto the biological activity of an antibody or ligand, e.g. the ability ofan antibody or ligand to elicit a cellular response. For example throughbinding to a target antigen, the antibody activates or suppresses a cellsignaling pathway, i.e. activates of inhibits the function of the targetantigen. For example, the antibody to be tested binds to a receptoractivating the NF-κB pathway and through this binding a response elementin the cell nucleus is activated. When linking this response element toa reporter gene, the activation can be easily monitored in the assay ofthe invention. The term “functionality” also includes the effectorfunctions of an antibody, e.g. C1q binding and complement dependentcytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); antibody-dependent cellularphagocytosis (ADCP), cytokine secretion, immune complex-mediated antigenuptake by antigen presenting cells; down regulation of cell surfacereceptors (e.g. B cell receptor); and B cell activation.

As used herein “target antigen” refers to any cell surface antigen thatcan be targeted by an antibody or fragment thereof. It also refers tothe receptor that can be targeted by a ligand. A “response element ofthe target antigen” refers to a specific transcription factor bindingelement, or cis acting element which can be activated or silenced onbinding of a certain transcription factor. In one embodiment theresponse element is a cis-acting enhancer element located upstream of aminimal promotor (e.g. a TATA box promotor) which drives expression ofthe reporter gene upon transcription factor binding.

As used herein “NF-κB” refers to the “nuclear factorkappa-light-chain-enhancer of activated B cells” and is a transcriptionfactor which is implicated in the regulation of many genes that code formediators of apoptosis, viral replication, tumorigenesis, variousautoimmune diseases and inflammatory responses. NFκB is present inalmost all eukaryotic cells. Generally, it is located in the cytosol inan inactive state, since if forms a complex with inhibitory kappa B(IκB) proteins. Through the binding of ligands to integral membranereceptors (also referred to as “receptors of the NF-κB pathway”, the IκBkinase (IKK) is activated. IKK is an enzyme complex which consists oftwo kinases and a regulatory subunit. This complex phosphorylates theIκB proteins, which leads to ubiquitination and therefore degradation ofthose proteins by the proteasome. Finally, the free NFκB is in an activestate, translocates to the nucleus and binds to the KB DNA elements andinduces transcription of target genes.

As used herein “NF-κB pathway” refers to the stimuli that lead tomodulation of activity of NF-κB. For example activation of the Toll-likereceptor signaling, TNF receptor signaling, T cell receptor and B cellreceptor signaling through either binding of a ligand or an antibodyresult in activation of NF-κB. Subsequently, phosphorylated NF-κB dimersbind to KB DNA elements and induce transcription of target genes.Exemplary KB DNA elements useful herein are referred to as “responseelement of the NF-κB pathway”. Hence, a “receptor of the NF-κB pathway”refers to a receptor which can trigger the modulation of activity ofNF-κB: Examples of a “receptor of the NF-κB pathway” are Toll-likereceptors, TNF receptors, T cell receptor and B cell receptor.Non-limiting examples of antibodies that upon binding to its targetresult in modulation of the activity of NF-κB are anti-CD40 antibodies,anti-DR5 antibodies, anti-DR4 antibodies, anti-41BB antibodies,anti-Ox40 antibodies and anti-GITR antibodies. Examples of ligands thatupon binding to its target result in modulation of the activity of NF-κBare OX40 ligand, 4-1BB ligand or CD40 ligand.

“High-throughput screening” as used herein shall be understood to meanthat a relatively large number of different antibody or ligandcandidates can be analyzed for binding and functionality with the novelassay described therein. Typical such high-throughput screening isperformed in multi-well microtiter plates, e.g. in a 96 well plate or a384 well plate or a plates with 1536 or 3456 wells.

The term “energy donor compound” refers to a fluorescent resonanceenergy transfer (FRET) energy donor compound, a bioluminescence energytransfer (BRET) energy donor compound and an AlphaScreen donor bead. Theterm “energy acceptor compound” refers to a fluorescent resonance energytransfer (FRET) energy acceptor compound, a bioluminescence energytransfer (BRET) energy acceptor compound and an AlphaScreen acceptorbead.

The term “FRET” refers to fluorescent resonance energy transferprocesses that occur between two chromophores. The chromophores as usedherein comprise, for example, fluorescent, luminescent and othernon-fluorescent components. “FRET,” “fluorescence resonance energytransfer,” “Förster resonance energy transfer” and “resonance energytransfer” are used interchangeably herein.

The term “time-resolved FRET” as used herein refers to energy transferprocesses that occur between two chromophores based on time-resolveddetection of the emission of the acceptor fluorophor (Morrison, L. E.,1988. Anal. Biochem., 174 (1) 101).

“FRET energy donor compound” also referred to as “FRET donor” or “FRETenergy donor” as used herein refers to a donor fluorophore useful inFRET, and are known in the art. Non-limiting examples of energy donorcompounds are listed in table B. Preferably the FRET energy donorcompound is a rare earth element, like for example Terbium cryptate(“Lumi4-Tb”) and Europium cryptates (Eu3+cryptate). Suitable lanthanidechelates useful in the method include those described for example inU.S. Pat. Nos. 5,622,821, 5,639,615, 5,656,433 and 4,822,733.

“FRET energy acceptor compound” also referred to as “FRET acceptor” or“FRET energy acceptor” as used herein refers to an acceptor fluorophoreuseful in FRET, and are known in the art, e.g. Alexa fluor dyes (Lifetechnologies) or Cy5, YFP, FITC, chemically modified allophycocyanine(XL665), d2. Further non-limiting examples of energy acceptor compoundsare listed in table B. Preferably the FRET energy acceptor compound isd2.

The term “BRET” refers to Resonance Energy Transfer (RET) between abioluminescent donor moiety (i.e. a BRET energy donor compound) and afluorescent acceptor moiety (i.e. a BRET energy acceptor compound).

The term “AlphaScreen” refers to Amplified Luminescent ProximityHomogeneous Assay and has been described e.g. in “Luminescent oxygenchanneling immunoassay: Measurement of particle binding kinetics bychemiluminescence.” Ullman, E F, et al. Proc. Natl. Acad. Sci. USA, Vol.91, pp. 5426-5430, June 1994.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody or ligand) and its binding partner (e.g., an antigen or areceptor). Unless indicated otherwise, as used herein, “bindingaffinity” refers to intrinsic binding affinity which reflects a 1:1interaction between members of a binding pair (e.g., antibody andantigen). The affinity of a molecule X for its partner Y can generallybe represented by the dissociation constant (Kd). Affinity can bemeasured by common methods known in the art, including those describedherein. Specific illustrative and exemplary embodiments for measuringbinding affinity are described in the following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

As used herein, the term “ligand” refers to any molecule that is able tobind to another molecule. Example of ligand molecules include, but arenot limited to peptides, proteins, carbohydrates, lipids, or nucleicacids. Preferred ligands to be analysed with the assay described hereinare peptides or proteins that are capable of binding to a targetantigen. Usually such target antigen is a cell surface receptor.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies, cross-Fab fragments; linear antibodies; single-chainantibody molecules (e.g. scFv); and multispecific antibodies formed fromantibody fragments. scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibodyfragments comprise single chain polypeptides having the characteristicsof a VH domain, namely being able to assemble together with a VL domain,or of a VL domain, namely being able to assemble together with a VHdomain to a functional antigen binding site and thereby providing theantigen binding property of full length antibodies.

As used herein, “Fab fragment” refers to an antibody fragment comprisinga light chain fragment comprising a VL domain and a constant domain of alight chain (CL), and a VH domain and a first constant domain (CH1) of aheavy chain.

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable region (VH), also called a variable heavy domain or a heavychain variable domain, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antigen binding domain” refers to the part of an antigenbinding molecule that comprises the area which specifically binds to andis complementary to part or all of an antigen. Where an antigen islarge, an antigen binding molecule may only bind to a particular part ofthe antigen, which part is termed an epitope. An antigen binding domainmay be provided by, for example, one or more antibody variable domains(also called antibody variable regions). Preferably, an antigen bindingdomain comprises an antibody light chain variable region (VL) and anantibody heavy chain variable region (VH).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species, usually prepared by recombinant DNAtechniques. Chimeric antibodies comprising a rabbit variable region anda human constant region are preferred. Other preferred forms of“chimeric antibodies” encompassed by the present invention are those inwhich the constant region has been modified or changed from that of theoriginal antibody to generate the properties according to the invention,especially in regard to C1q binding and/or Fc receptor (FcR) binding.Such chimeric antibodies are also referred to as “class-switchedantibodies”. Chimeric antibodies are the product of expressedimmunoglobulin genes comprising DNA segments encoding immunoglobulinvariable regions and DNA segments encoding immunoglobulin constantregions. Methods for producing chimeric antibodies involve conventionalrecombinant DNA and gene transfection techniques are well known in theart. See e.g. Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependentcellular phagocytosis (ADCP), cytokine secretion, immunecomplex-mediated antigen uptake by antigen presenting cells; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Particularamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G₃₂₉, P329G, or Pro329Gly.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, theC-terminal lysine (Lys447) of the Fc region may or may not be present.Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991. A “subunit”of an Fc domain as used herein refers to one of the two polypeptidesforming the dimeric Fc domain, i.e. a polypeptide comprising C-terminalconstant regions of an immunoglobulin heavy chain, capable of stableself-association. For example, a subunit of an IgG Fc domain comprisesan IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. As also mentioned forchimeric and humanized antibodies according to the invention the term“human antibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation.)

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

The term “hypervariable region” or “HVR,” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) Hypervariable regions(HVRs) are also referred to as complementarity determining regions(CDRs), and these terms are used herein interchangeably in reference toportions of the variable region that form the antigen binding regions.This particular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917(1987), where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table A as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. CDRs also comprise“specificity determining residues,” or “SDRs,” which are residues thatcontact antigen. SDRs are contained within regions of the CDRs calledabbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2,a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633(2008).) Unless otherwise indicated, HVR residues and other residues inthe variable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“No substantial cross-reactivity” means that a molecule (e.g., anantibody) does not recognize or specifically bind an antigen differentfrom the actual target antigen of the molecule (e.g. an antigen closelyrelated to the target antigen), particularly when compared to thattarget antigen. For example, an antibody may bind less than about 10% toless than about 5% to an antigen different from the actual targetantigen, or may bind said antigen different from the actual targetantigen at an amount consisting of less than about 10%, 9%, 8% 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%,1%, or 0.5%, and most preferably less than about 0.2% or 0.1% antigendifferent from the actual target antigen.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W. H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “antigen-binding site of an antibody” when used herein refer tothe amino acid residues of an antibody which are responsible forantigen-binding. The antigen-binding portion of an antibody comprisesamino acid residues from the “complementary determining regions” or“CDRs”. “Framework” or “FR” regions are those variable domain regionsother than the hypervariable region residues as herein defined.Therefore, the light and heavy chain variable domains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding and defines the antibody'sproperties. CDR and FR regions are determined according to the standarddefinition of Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991) and/or those residues from a “hypervariable loop”.

Antibody specificity refers to selective recognition of the antibody fora particular epitope of an antigen. Natural antibodies, for example, aremonospecific.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen.

The term “bispecific” antibody as used herein denotes an antibody thathas at least two binding sites each of which bind to different epitopesof the same antigen or a different antigen. Techniques for makingmultispecific antibodies include, but are not limited to, recombinantco-expression of two immunoglobulin heavy chain-light chain pairs havingdifferent specificities (see Milstein and Cuello, Nature 305: 537(1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)),and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168).Multi-specific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (WO2009/089004); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81(1985)); using leucine zippers to produce bi-specific antibodies (see,e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using“diabody” technology for making bispecific antibody fragments (see,e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber etal., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodiesas described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” (see, US 2008/0069820, for example).

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in an antibody molecule.As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denotethe presence of two binding sites, four binding sites, and six bindingsites, respectively, in an antibody molecule. The bispecific antibodiesaccording to the invention are at least “bivalent” and may be“trivalent” or “multivalent” (e.g. “tetravalent” or “hexavalent”).

Antibodies of the present invention have two or more binding sites andare bispecific. That is, the antibodies may be bispecific even in caseswhere there are more than two binding sites (i.e. that the antibody istrivalent or multivalent). Bispecific antibodies of the inventioninclude, for example, multivalent single chain antibodies, diabodies andtriabodies, as well as antibodies having the constant domain structureof full length antibodies to which further antigen-binding sites (e.g.,single chain Fv, a VH domain and/or a VL domain, Fab, or (Fab)2) arelinked via one or more peptide-linkers. The antibodies can be fulllength from a single species, or be chimerized or humanized.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transfectants” and “transfected cells” include theprimary subject cell and cultures derived there from without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody or ligand) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen/ligand andreceptor). The affinity of a molecule X for its partner Y can generallybe represented by the dissociation constant (Kd). Affinity can bemeasured by common methods known in the art, including those describedherein. Specific illustrative and exemplary embodiments for measuringbinding affinity are described in the following.

As used herein, the term “binding” or “specifically binding” refers tothe binding of the antibody to an epitope of the antigen or the bindingof a ligand to a receptor in an in-vitro assay, preferably byfluorescence resonance energy transfer (FRET).

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody.

As used herein, the terms “engineer, engineered, engineering,”particularly with the prefix “glyco-,” as well as the term“glycosylation engineering” are considered to include any manipulationof the glycosylation pattern of a naturally occurring or recombinantpolypeptide or fragment thereof. Glycosylation engineering includesmetabolic engineering of the glycosylation machinery of a cell,including genetic manipulations of the oligosaccharide synthesispathways to achieve altered glycosylation of glycoproteins expressed incells. Furthermore, glycosylation engineering includes the effects ofmutations and cell environment on glycosylation. In one embodiment, theglycosylation engineering is an alteration in glycosyltransferaseactivity. In a particular embodiment, the engineering results in alteredglucosaminyltransferase activity and/or fucosyltransferase activity.

II. Novel Assay

The inventors developed an assay that is suitable for a high-throughputformat (384 well) which enables combined analysis of antibody or ligandbinding and functionality in one well or vial. Functionality of theantibody or ligand (e.g. the biological activity of an antibody orligand, e.g. the ability of an antibody or ligand to elicit a cellularresponse, for example to activate or inhibit the target antigen) isevaluated by using transfected reporter cell lines which have a reportergene expressed upon activation of a response element. In one embodimentsaid reporter gene is selected from a gene encoding for a fluorescentprotein (e.g. green fluorescent protein, GFP) and/or a gene encoding foran enzyme whose catalytic activity can be detected (e.g. Luciferase). Toaddress the binding, a cell-based FRET method, BRET or AlphaScreen areapplied.

In one embodiment there is provided an in vitro assay for determiningthe binding and functionality of an antibody or a ligand specificallybinding to a target antigen comprising the following steps

i) providing cells which

-   -   a) express the target antigen on their surface,    -   b) are covalently or noncovalently labelled with an energy donor        compound and    -   c) comprise a reporter gene under the control of a response        element of the target antigen

ii) adding the antibody or ligand to be tested

iii) measuring the binding to the target antigen by determining theenergy transfer, wherein the energy acceptor compound is covalently ornoncovalently conjugated either to the antibody to be tested or to asecondary antibody binding to the first antibody; and

iv) determining functionality of the antibody or ligand by correlatingthe level of the expression of the reporter gene with the level oftarget antigen activation or inhibition.

In one embodiment the energy donor and acceptor compound are afluorescent resonance energy transfer (FRET) energy donor and acceptorcompound and the energy transfer determined in step iii) is fluorescentresonance energy transfer (FRET). In one embodiment the FRET is timeresolved FRET.

A technology to study protein-protein interactions in a microplateformat is the time-resolved FRET. Time-resolved fluorescence resonanceenergy transfer (TR-FRET) applications are based on energy transferbetween a donor and an acceptor molecule. FRET is a non-radiative energytransfer from a fluorescent donor molecule to an appropriate acceptormolecule. When a donor molecule is excited by a light source, itproduces fluorescence. If an acceptor molecule is in close proximity,and the emission spectrum of the donor molecule overlaps with theexcitation spectrum of the acceptor molecule, the donor molecule,instead of emitting fluorescent light, can transfer its excitationenergy to the acceptor molecule. The acceptor molecule will then emitfluorescence at the acceptor emission wavelength. The apparition offluorescent light at the acceptor emission wavelength indicates that thedonor and acceptor molecules are in close proximity to each other as thedonor and acceptor molecules need to be less than about 20 nm apart forenergy transfer to occur, e.g., 5-10 nm apart. Typically, closeproximity between the donor and acceptor molecules is achieved viabioaffinity interactions, e.g., protein-protein binding,antigen-antibody binding, ligand-receptor binding, DNA hybridization,and DNA-protein binding.

A large variety of donor and acceptor molecules exist. Typically thedonor and acceptor molecules used in FRET assays are fluorophores thathave short half-lives. The performance of traditional FRET chemistriescan be reduced by background fluorescence from sample components such asbuffers, proteins, chemical compounds, and cell lysate. Detectedfluorescence intensities must be corrected for this auto-fluorescence,which reduces assay sensitivity and can complicate resultinterpretation. This type of background fluorescence is transient (witha lifetime in the nanosecond range) and can therefore be eliminatedusing time-resolved methodologies.

TR-FRET takes advantage of the unique properties of lanthanide ions suchas europium (Eu), terbium (Tb), samarium (Sm), and dysprosium (Dy) ions.Because of their specific photophysical and spectral properties,complexes of rare earth ions are of interest for fluorescenceapplications in biology. Specifically, they have large Stoke's shiftsand long emission half-lives (from μsec to msec) when compared to moretraditional fluorophores.

FRET energy acceptors are commercially available at PerkinElmer (e.g.,LANCE® products), Invitrogen (e.g., LanthaScreen® products), and CisbioBioassays (e.g., HTRF® products).

In some embodiments, the FRET energy donor and the FRET energy acceptormay be chosen based upon one or more of the fluorophores listed in thetable below.

TABLE B Examples of FRET energy donor and energy acceptor moleculesFLUOROPHORE Excitation (nm) Emission (nm) 5-Carboxynapthofluorescein512/598 563/668 5-ROX (carboxy-X-rhodamine) 578 604 567 591 Alexa Fluor568 ™ 577 603 Alexa Fluor 594 ™ 590 617 594 618 Alexa Fluor 633 ™ 632650 Alexa Fluor 647 ™ 647 666 Alexa Fluor 660 ™ 668 698 Alexa Fluor680 ™ 679 702 Allophycocyanin (APC) 630-645 655-665 APC-Cy7 625-650 755BOBO ™ -3 570 602 Bodipy 492-591 509-676 Bodipy TR 589 617 Bodipy TR ATP591 620 Calcium Crimson ™ 588 611 589 615 Carboxy-X-rhodamine (5-ROX)576 601 Cy3.5 ™ 581 598 Cy5.1 8 649 666 Cy5.5 ™ 675 695 Cy5 ™ 649 666Cy7 ™ 710, 743 767, 805 710 767 743 805 Dysprosium 305-335 465-495565-595 Europium 315-350 600-635 675-715 Europium (III) chloride 315-350600-635 675-715 FL-645 615-625 665 Fura Red ™ (high pH) 572 657 LaserPro795 812 Samarium 325-355 475-505 545-575 585-615 630-660 SureLight ®640-660 660-680 Terbium 305-335 475-505 530-560 570-600 605-635 TexasRed ™ 595 620 Texas Red-X ™ conjugate 595 615 Thiadicarbocyanine (DiSC3)651 674 653 675 Thiazine Red R 596 615 TO-PRO-1 515 531 TO-PRO-3 644 657TO-PRO-5 747 770 TOTO-3 642 660 ULight ® 630-655 655-675 Ultralite 656678 X-Rhodamine 580 605 XRITC 582 601 YO-PRO-3 613 629

The following information may also be considered when selecting a FRETenergy donor and FRET energy acceptor combination. U.S. Pat. No.5,998,146, herein incorporated by reference, describes the use oflanthanide chelate complexes, in particular of europium and terbiumcomplexes combined with fluorophores or quenchers. It also describesproperties of the long-lived lanthanide chelate complexes.

FRET systems based on metallic complexes as energy donors and dyes fromthe class of phycobiliproteins as energy acceptors are known in the art(EP 76 695; Hemmilae, Chemical Analysis 117, John Wiley & Sons, Inc.,(1991) 135-139). Established commercial systems (e.g., from Wallac, OYor Cis Bio Packard) use a FRET pair consisting of a lanthanide chelateas the metallic complex and a phycobiliprotein.

The properties of the lanthanide-chelate complexes in particular ofeuropium or terbium complexes are known and can be used in combinationwith quenchers as well as in combination with fluorophores.

Ruthenium complexes per se are used as fluorophores or luminophoresespecially for electro-chemoluminescence. Ruthenium-chelate complexesare, for example, known from EP 178 450 and EP 772 616 in which methodsfor coupling these complexes to biomolecules are also described. Theiruse as energy donors in FRET systems is not discussed there.

Allophycocyanins have properties such as unusually high extinctioncoefficients (about 700000 L/M cm) and also extremely high emissioncoefficients. These are useful prerequisites for their use asfluorophore acceptors in FRET systems. Moreover these dyes are known tobe readily soluble in water and stable.

The term low molecular fluorophore refers to fluorophoric dyes having amolecular weight between 300 and 3000 Da. Such low molecularfluorophoric groups such as xanthenes, cyanins, rhodamines and oxazineshave considerable disadvantages compared to the APCs with regard toimportant characteristics. Thus for example their extinctioncoefficients are substantially lower and are in the range of ca. 100000L/M cm.

In some embodiments, the methods and assays of the invention make use ofhomogeneous TR-FRET assay techniques. TR-FRET is a combination oftime-resolved fluorescence (TRF) and FRET. TRF reduces backgroundfluorescence by delaying reading the fluorescent signal, for example, byabout 10 nano seconds. Following this delay (i.e., the gating period),the longer lasting fluorescence in the sample is measured. Thus, usingTR-FRET, interfering background fluorescence, that may for example bedue to interfering substances in the sample, is not co-detected, butrather, only the fluorescence generated or suppressed by the energytransfer is measured. The resulting fluorescence of the TR-FRET systemis determined by means of appropriate measuring devices. Suchtime-resolved detection systems use, for example, pulsed laser diodes,light emitting diodes (LEDs) or pulsed dye lasers as the excitationlight source. The measurement occurs after an appropriate time delay,i.e., after the interfering background signals have decayed. Devices andmethods for determining time-resolved FRET signals are described in theart.

This technique requires that the signal of interest must correspond to acompound with a long fluorescent lifetime. Such long-lived fluorescentcompounds are the rare earth lanthanides. For example, Eu3+ has afluorescent lifetime in the order of milliseconds. TR-FRET requires aFRET energy donor and a FRET energy acceptor, as described above. Aswith FRET, a TR-FRET energy donor and acceptor pair can be selectedbased on one or more, including all, of the following: (1) the emissionspectrum of the FRET energy donor should overlap with the excitationspectrum of the FRET energy acceptor; (2) The emission spectra of theFRET partners (i.e., the FRET energy donor and the FRET energy acceptor)should show non-overlapping fluorescence; (3) the FRET quantum yield(i.e., the energy transferred from the FRET donor to the FRET acceptor)should be as high as possible (for example, FRET should have about a1-100%, e.g., a 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, and99% efficiency over a measured distance, of 1-20 nm, e.g., 5-10 nm); (4)the FRET signal (i.e., fluorescence) must be distinguishable fromfluorescence produced by the sample, e.g., autofluorescence; and (5) theFRET donor and the FRET acceptor should have half lives that allowdetection of the FRET signal (e.g., FRET can be bright and can occur ona timescale ranging from 10-9 seconds to 10-4 seconds).

In some embodiments, the TR-FRET donor and the TR-FRET acceptor may bechosen based upon one or more of the fluorophores listed in the tableabove.

In some embodiments, the TR-FRET donor may be a lanthanide ion, e.g., alanthanide ion bound to a chelate. In some embodiments, the lanthanideion may be a europium, terbium, samarium, or dysprosium ion. As usedherein, Eu includes Eu and all Eu ions, e.g., Eu3+. In some embodiments,the TR-FRET donor may be DsRed. In some embodiments, the TR-FRET donormay be Ri2. It is to be understood that selection of the appropriateTR-FRET donor requires consideration of the above listed criteria andthe specific TR-FRET acceptor selected.

In some embodiments, the TR-FRET acceptor may be selected from the groupconsisting of fluorescein, Cy5, allophycocyanin (APC—e.g., XL665, d2(Cisbio), and BG-647), and a fluorescent protein (e.g., GFP, CFP, YFP,BFP, RFP, and other GFP variants).

In some embodiments, the TR-FRET donor may be terbium and the TR-FRETacceptor may be fluorescein. In some embodiments, the TR-FRET donor maybe Eu and the TR-FRET acceptor may be Cy5 or APC (e.g., XL665, d2(Cisbio), BG-647, and Cy5-related TR-FRET acceptors). In one embodimentthe FRET energy donor compound is Terbium cryptate and/or the FRETenergy acceptor compound is d2.

In some embodiments, the TR-FRET donor and the TR-FRET acceptor may becombined with a second compound that enhances the function of theTR-FRET donor and/or the TR-FRET acceptor. For example, the TR-FRETdonor and the TR-FRET acceptor may be combined with cryptateencapsulation to extend the half-life of the fluorophore. Alternatively,or in addition, the TR-FRET donor the TR-FRET acceptor may be combinedwith, e.g., DELFIA® enhancement system. In some embodiments, the TR-FRETdonor and the TR-FRET acceptor may be combined with, for example,buffers, salts, enhancers, chelators, and stabilizers (e.g.,photo-stabilizers) that enhance or extend the life or detection of theTR-FRET signal.

Molecules, e.g., proteins, may be labeled directly or indirectly withsuitable FRET or TR-FRET donors and acceptors, as described above anddescribed in U.S. Pat. Nos. 4,925,804, 5,637,509, 4,761,481, 4,920,195,5,032,677, 5,202,423, 5,324,825, 5,457,186, 5,571,897, 7,250,517,US2005/0202565, which are herein incorporated by reference.

Time-resolved FRET is measured in a time-resolved manner by introducinga time delay of approximately 50-150 μs between excitation of the donorand emission measurement of the acceptor. Thus, short-lived backgroundfluorescence generated by the medium, the biological preparation or thedirect excitation of the acceptor are not measured and assay sensitivityis increased. A commonly used acceptor molecule is an organic redfluorescent dye called d2 which is 100 times smaller than XL665 withexhibiting photophysical properties very similar to XL665. (Amoravain M,Lyotard S, Jaga D, Lebreton M L, Servent F, Bomer U. Introduction of anew HTRF acceptor, d2: Development of a complete GPCR platform for a Gs,Gi and Gq screening. SBS; 11th Annual Conference; Geneva. 2005.).

Any of a variety of light-emitting and light-detecting instruments canbe used to initiate FRET (e.g., excite a FRET donor or excite a reagentcapable of exciting the FRET donor) and/or detect an emission producedfrom said FRET. The light emissions produced by the first and secondmember of the FRET pair as a result of the above methodologies can bedetected or measured visually, photographically, actinometrically,spectrophotometrically, or by any other convenient means to determinethe amount thereof, which is related to the amount of each component inthe medium.

The binding of the antibody or ligand to the target antigen can bedetermined qualitatively, i.e. by the presence or absence of the FRETsignal; with the absence of any FRET signal being indicative of nobinding. Usually the “absence of a FRET signal” is defined by a certainthreshold, i.e. after deduction of any background signal. The backgroundsignal is usually determined by performing the FRET assay with allreagents but the antibody or ligand to be tested. The binding of theantibody or ligand to the target antigen can be determined quantitavely,i.e. the level or strength of binding can be determined with the FRETmethod. Towards this end the antibody or ligand to be tested is testedin different concentrations and the half maximal effective concentration(EC50) is determined. EC50 refers to the concentration of the antibodyor ligand at which the antibody or ligand binds halfway between thebaseline and maximum after a specified exposure time. The EC50 of thedose response curve therefore represents the concentration of theantibody or ligand where 50% of its maximal binding is observed. The KD(dissociation constant) can be calculated from the dose response curveby methods known in the art.

In one embodiment the binding of the antibody or ligand to the targetantigen is determined with bioluminescence resonance energy transfer(BRET). Accordingly in one embodiment the energy donor and acceptorcompound are a bioluminescence energy transfer (BRET) energy donor andacceptor compound and the energy transfer determined in step iii) isbioluminescence energy transfer (BRET). The BRET assay technology isbased on the efficient Resonance Energy Transfer (RET) between abioluminescent donor moiety and a fluorescent acceptor moiety. BRET is anaturally occurring phenomenon and differs from FRET in that it uses aluciferase as the donor. In one embodiment a luciferase (Rluc) isolatedfrom the sea pansy Renilla reniformis and a coelenterazine substratenamed DeepBlueC (DBC) are used as the donor. In the presence of oxygen,Rluc catalyzes the transformation of DBC into coelenteramide withconcomitant light emission peaking at 395 nm (blue light). When asuitable acceptor is in close proximity, the blue light energy iscaptured by RET. In one embodiment the acceptor in BRET is a GFP variant(GFP2) that is engineered to maximally absorb the energy emitted by theRluc/DBC reaction. Excitation of GFP2 by RET results in an emission ofgreen light at 510 nm. Energy transfer efficiencies between Rluc/DBC andGFP2 are determined ratiometrically by dividing the acceptor emissionintensity by the donor emission intensity. This ratiometric measurementis referred to as the BRET signal and reflects the proximity of Rluc toGFP. In another embodiment Rluc is used as the donor, the derivative ofcoelenterazine as its substrate and a yellow fluorescent protein (YFP)as the acceptor.

In one embodiment the binding of the antibody or ligand to the targetantigen is determined with an AlphaScreen (Amplified LuminescentProximity Homogeneous Assay). Accordingly in one embodiment the energydonor and acceptor compound are an alpha screen acceptor and donor beadand the energy transfer determined in step iii) is an energy transferfrom a singlet oxygen to an thioxene derivative within the acceptorbead. AlphaScreen is a non-radioactive, homogeneous proximity assayusing an “acceptor” and a “donor” bead. AlphaScreen is a bead-baseddetection system used to study biomolecular interactions in a microplateformat. Binding of molecules captured on the beads leads to an energytransfer from one bead to the other, ultimately producing aluminescent/fluorescent signal. Every AlphaScreen assay contains twobead types, donor beads and acceptor beads. Both bead types are coatedwith a hydrogel which minimizes nonspecific binding andself-aggregation, and provides reactive aldehyde groups for conjugatingbiomolecules to the bead surface.

Every AlphaScreen assay contains two bead types, donor beads andacceptor beads. Each bead type contains a different combination ofchemicals, which are key elements of the AlphaScreen technology. Donorbeads contain a photosensitizer, phthalocyanine, which converts ambientoxygen to an excited form of singlet oxygen, upon laser illumination at680 nm Like other excited molecules, singlet oxygen has a limitedlifetime prior to falling back to ground state. Within its 4 sechalf-life, singlet oxygen can diffuse approximately 200 nm in solution.If an acceptor bead is within that proximity, an energy transfer from asinglet oxygen to an thioxene derivative within the acceptor bead takesplace, subsequently culminating in light production at 520-620 nm. Inthe absence of an acceptor bead, singlet oxygen falls to ground stateand no signal is generated. In one embodiment the cells used in theassay are labelled with biotinilated WGA bound to streptavidin coateddonor beads. In one embodiment the acceptor beads are conjugated toProtein A. The antibody to be tested can easily be labeled with theseacceptor beads as Protein A binds to the Fc domain of the antibody.

Methods for labeling the cell surface and the antibody or ligand to betested or secondary antibody for BRET, FRET or AlphaScreen are known inthe art. For example, binding partners can be labeled directly orindirectly (e.g., via a tag like non-peptide tags such as biotin,digoxigenin, fluorescein, dinitrophenol and also peptidic tags such asFLAG, HisTag, cmyc, HA, V5, streptag, ACP/MCP Tag or usingavidin-streptavidin interactions), for example as described by Yang etal., Analytical Biochemistry 351:158-160, 2006, which is hereinincorporated by reference. ACP-tag and MCP-tag are small protein tagsbased on the acyl carrier protein. The presence of an added synthase isrequired for the formation of a covalent link between the ACP-tag orMCP-tag and their substrates, which are derivatives of Coenzyme A. Inthe labeling reaction, the group conjugated to CoA is covalentlyattached to the ACP-tag or MCP-tag by a recombinant synthase. Labels canbe covalently attached to a tag using either ACP-Synthase (NEB #P9301)for ACP-tag labeling or SFP-Synthase (NEB #P9302) for dual ACP- andMCP-tag labeling.

In one embodiment the target antigen is covalently or noncovalentlylabelled with the energy donor compound. For example, the protein to beanalysed (e.g. the target antigen of the antibody or ligand to beanalysed herein) can be labeled by linking it to the 20 kDa DNA repairenzyme human O⁶-alkylguanine-DNA-alkyltransferase (AGT). This enzyme canbuild a covalent bond to a O⁶-benzylguanine (BG) derivative which itselfis coupled to the donor or acceptor fluorescent dye. For the generationof cells expressing the labeled target antigen of interest, the cellsare transfected with a plasmid encoding the humanO⁶-alkylguanine-DNA-alkyltransferase (AGT) fused to the protein ofinterest. Following transfection, the fusion protein expressed at thecell surface is labeled using BG-fluorophore and can then be used forFRET based interaction studies. Preferably the target antigen is labeledwith the FRET donor molecule.

The antibody or ligand to be analysed (which binds to the labelledtarget antigen expressed at the cell surface) can be covalently ornoncovalently labeled with a energy donor or acceptor compound, e.g. afluorescent dye or an AlphaScreen bead. It is also possible to label asecondary antibody that binds to the antibody or ligand to be analysed(e.g. anti-human IgG).

Labelling can also be achieved through wheat germ agglutinin (WGA)labelled with a FRET donor or acceptor molecule. Hence in one embodimentthe energy donor compound is covalently or noncovalently linked to wheatgerm agglutinin (WGA). Thus, instead of transfecting the cells andlabeling the fusion proteins as described above, the cells themselvescan be labeled with the donor molecule. WGA binds with high affinity tocarbohydrates such as N-acetyl glucosamine (GlcNAc) andN-acetylneuraminic acid (also called sialic acid). Due to theomnipresence of such sugar types on the cell surface, binding of WGAconjugated with a donor or acceptor fluorophor allows random labeling ofthe cell surface. Interaction of an antibody or ligand binding to theantigen of interest embedded in the cell membrane can then be detectedby an energy transfer from the WGA-Terbium to the acceptor label coupleddirectly to the antibody or ligand to be evaluated or to a secondarydetection antibody.

In one embodiment the reporter gene is selected from a gene coding for afluorescent protein or a gene coding for an enzyme whose catalyticactivity can be detected. In one embodiment the fluorescent protein isselected from the group consisting of green fluorescent protein (GFP),yellow fluorescent protein (YFP), red fluorescent protein (RFP), Bluefluorescent protein (BFP, Heim et al. 1994, 1996), a cyan fluorescentvariant known as CFP (Heim et al. 1996; Tsien 1998); a yellowfluorescent variant known as YFP (Oruro et al. 1996; Wachter et al.1998); a violet-excitable green fluorescent variant known as Sapphire(Tsien 1998; Zapata-Hommer et al. 2003); and a cyan-excitable greenfluorescing variant known as enhanced green fluorescent protein or EGFP(Yang et al. 1996) enhanced green fluorescent protein (EGFP) and can bemeasured e.g. by live cell imaging (e.g. Incucyte) or fluorescentspectrophotometry. In one embodiment the enzyme whose catalytic activitycan be detected is selected from the group consisting of luciferase,beta Galactosidase, Alkaline Phosphatase. In one preferred embodimentthe reporter gene is encoding for GFP.

In one embodiment the reporter gene encodes for luciferase. The activityof luciferase can be detected by commercially available assays, e.g. byLuciferase 1000 Assay System (or ONE-Glo™ Luciferase Assay System (bothPromega). The Luciferase 1000 Assay System contains coenzyme A (CoA)besides luciferin as a substrate, resulting in a strong light intensitylasting for at least one minute. For assaying the intracellularluciferase, it is necessary to lyse the cells prior to detection.Therefore, a cell lysis buffer was provided separately to the Luciferase1000 assay system. In comparison, the ONE-Glo™ Luciferase Assay Systemcombines the Luciferase substrate with a cell lysis reagent and alsoshows a more stable signal. The light which is produced as a by-productof the reaction is collected by the luminometer from the entire visiblespectrum. In the examples shown herein the signal was proportional tothe amount of produced luciferase and therefore proportional to thestrength of the activation of the NFκB promotor. In another embodiment aLuciferase assay is used wherein the luciferase is secreted from thecells. Hence the assay can be performed without lysis of the cells.

The expression of the reporter gene can be directly correlated with thefunctionality of the ligand or antibody to be tested. For example whenusing a gene encoding for a fluorescent protein or a gene encoding forluciferase as a reporter gene, the amount of light detected from thecells correlates directly with the target antigen activation orinhibition of the antibody or ligand to be tested.

In one embodiment the antibody or ligand are tested in differentconcentrations and the half maximal effective concentration (EC50) ofreporter gene activation or inhibition is determined. EC50 refers to theconcentration of the antibody or ligand at which the antibody or ligandactivates or inhibits the reporter gene halfway between the baseline andmaximum after a specified exposure time. The EC50 of the dose responsecurve therefore represents the concentration of the antibody or ligandwhere 50% of its maximal activating or inhibitory effect on the targetantigen is observed.

In one embodiment the cells naturally express the target antigen. In oneembodiment the target antigen is a cell surface receptor. In anotherembodiment the cells are expressing the target antigen after beingtransfected with a gene copy encoding for said target antigen. Inanother embodiment cells naturally expressing the target antigen aretransfected with an additional gene copy encoding for said targetantigen. In one embodiment the cells are transfected with a gene copyencoding for the target antigen fused to a gene copy encoding for thehuman O⁶-alkylguanine-DNA-alkyltransferase (AGT). In one embodiment thecells are eukaryotic cells, preferably human or primate cells.

In one embodiment the binding is measured as a dilution curve fordetermination of KD and EC50 values as outlined above.

The binding of the antibody or ligand to the target antigen elicits acellular response which results in a modulation of the activity of theresponse element, either directly or through a cascade of cellsignalling. The response element is a DNA element which can be silencedor activated by transcription factors or the like. Response elements areknown in the art and are commercially available e.g. in reportervectors. Usually the response element comprises DNA repeat elements andis a cis-acting enhancer element located upstream of a minimal promotorwhich drives expression of a reporter gene upon transcription factorbinding. Examples for response elements and their transcription factorsuseful herein are mentioned in the below table:

Transcription factor/Response element Description AP1(1) Monitoringinduction of the activator protein 1(AP) and the stress-activatedprotein kinase/Jun N-terminal kinase (SAPK/JNK) signal transductionpathway. AP1(2) Monitoring the induction of the protein kinase C (PKC)signal transduction pathway, as well as related pathways such as theMAPK pathway. AP3 Measuring transcriptional activity of activatorprotein 3. AR Measuring transcriptional activity of androgen receptor.The androgen receptor functions as a steroid-hormone activatedtranscription factor. Upon binding the hormone ligand, the receptordissociates from accessory proteins, translocates into the nucleus,dimerizes, and then stimulates transcription of androgen responsivegenes. CRE(1) Measuring transcriptional activity of cAMP binding protein(CREB). Several signal transduction pathways are associated with thecAMP response element (CRE), including Jun N-terminal kinase (JNK), p38,and protein kinase A (PKA). Induction of these pathways enablesendogenous transcription factors, such as CREB or ATF, to bind CRE.E2F(1) Measuring transcriptional activity of E2F transcription factorfamily, including E2F1, E2F2, E2F3, E2F4, E2F5. The E2F protein familyplays a crucial role in the control of cell cycle and action of tumorsuppressor proteins and is also a target of the transforming proteins ofsmall DNA tumor viruses.These proteins bind preferentially toretinoblastoma protein pRB and mediate both cell proliferation andp53-dependent/independent apoptosis. ELK1 Measuring transcriptionalactivity of ELK1. ELK1 is a member of the Ets family of transcriptionfactors and of the ternary complex factor (TCF) subfamily. Proteins ofthe TCF subfamily form a ternary complex by binding to the serumresponse factor and the serum reponse element in the promoter of thec-fos proto-oncogene. ELK1is a nuclear target for the ras- raf-MAPKsignaling cascade. ER Measuring the induction of the estrogen responseelement (ERE). Binding of the activated estrogen receptor to thecis-acting ERE enhancer element induces transcription and activates theluciferase reporter gene. GAS Monitoring the induction of STAT1, acomponent of JAK/STAT-mediated (interferon- signal transductionpathways. Cytokines bind and induce receptor gamma dimerization at thecell surface, causing the receptor itself to be activationphosphorylated. The phosphorylated receptor then acts as a docking sitefor sequence) STAT1. STAT1 is phosphorylated, dimerizes and translocatesto the nucleus to regulate transcription. GATA Measuring transcriptionalactivity of globin transcription factor (GATA) family. The GATA familyof transcription factors contains six zinc-finger binding proteins thatregulate differentiation and cell proliferation. GATA family members areinvolved in hematopoietic, cardiac and gut development. GR Monitoringthe induction of the glucocorticoid response element (GRE) and theglucocorticoid-mediated signaling transduction pathway. HIF-1 Measuringtranscriptional activity of hypoxia inducible factor-1 (HIF-1). HIF-1binds to the hypoxia-response element and activates genes involved inangiogenesis, glucose metabolis, cell proliferation/survival andinvasion/metastasis. HSE Monitoring the activation of heat shock factor(HSF) and heat shock- mediated signal transduction pathways. IRF-1Measuring transcriptional activity of interferon regulatory factor 1.IRF1 is a member of the interferon regulatory transcription factor (IRF)family. IRF1 serves as an activator of interferons alpha and betatranscription, and in mouse it has been shown to be required fordouble-stranded RNA induction of these genes. ISRE Monitoring theinduction of the STAT1 and STAT2 components of Jak/STAT-mediated signaltransduction pathways. Signaling molecules, including type I (IFN-a and-b) and type II (IFN-g) interferons, induce signaling by bindingreceptors and causing receptor dimerization at the cell surface. Thisdimerization causes the receptor itself to be phosphorylated and act asa docking site for transcription factors, including STAT1 and STAT2. TheSTAT proteins are then phosphorylated, dimerize and translocate to thenucleus, where the STAT1 and STAT2 heterodimer regulates transcriptionby binding to the IFN-stimulated response element (ISRE). MEF-1Measuring transcriptional activity of myogenic factor 3 (MYOD1). MEF-2Measuring transcriptional activity MADS box transcription enhancerfactor 2A, 2B, 2C and 2D. MEF-3 Monitoring the activation of myelin geneexpression factor 3. NFAT Monitoring the induction of nuclear factor ofactivated T-cells (NFAT)- mediated signal transduction pathways. Severalpathways are associated with the NFAT enhancer element, includingcalcineurin and protein kinase C. NFκB Monitoring the activation of thenuclear factor of kappa light polypeptide gene enhancer in B-cells(NFκB) signal transduction pathway. NFκB is a transcription regulatorthat is activated by various intra- and extra-cellular stimuli such ascytokines, oxidant-free radicals, ultraviolet irradiation, and bacterialor viral products. Activated NFκB translocates into the nucleus andstimulates the expression of genes involved in a wide variety ofbiological functions. p53 Monitoring p53-mediated signal transductionpathways. p53 is a tumor suppressor that plays a crucial role in anumber of cellular processes, including the suppression of cellproliferation after DNA damage. PR Monitoring the induction ofprogesterone receptor. RAR Monitoring the induction of the retinoic acidresponse element (RARE). RXR Monitoring the activation of retinoid Xreceptors (RXR) and RXR-mediated signal transduction pathway. Retinoid Xreceptors (RXRs) and retinoic acid receptors (RARs) are nuclearreceptors that mediate the biological effects of retinoids by theirinvolvement in retinoic acid-mediated gene activation. These receptorsexert their action by binding, as homodimers or heterodimers, tospecific sequences in the promoters of target genes and regulating theirtranscription. Smad Measuring transcriptional activity of a family ofMad-related transcription factors. Sp1 Measuring transcriptionalactivity of Sp1. Sp1 is a sequence-specific transcription factor thatrecognizes 5′-GGGGCGGGGC-3′ and closely related sequences, which areoften referred to as GC boxes. Sp1 was initially identified as a HeLacell derived factor that selectively activates in vitro transcriptionfrom the SV40 promoter and binds to the multiple GC boxes in the 21-bprepeated elements in SV40. Sp1 has been described as a ubiquitoustranscription factor that is required for the constitutive and inducibleexpression of a variety of genes, such as in cell cycle or mammaliandevelopment. SRE Monitoring the induction of the serum response element(SRE) and the mitogen-activated protein (MAP) kinase signal transductionpathway. SRF Monitoring the induction of serum response factor (c-fosserum response element-binding transcription factor). Stat1 p84/p91Measuring transcriptional activity of signal transducer and activator oftranscription 1. Stat1 is a member of the STAT protein family. Inresponse to cytokines and growth factors, STAT family members arephosphorylated by the receptor associated kinases, and then form homo-or heterodimers that translocate to the cell nucleus where they act astranscription activators. This protein can be activated by variousligands including interferon-alpha, interferon-gamma, EGF, PDGF andIL-6. Stat4 Measuring transcriptional activity of signal transducer andactivator of transcription 4. Stat4 protein encoded by this gene is amember of the STAT family of transcription factors. In response tocytokines and growth factors, STAT family members are phosphorylated bythe receptor associated kinases, and then form homo- or heterodimersthat translocate to the cell nucleus where they act as transcriptionactivators. This protein is essential for mediating responses to IL-12in lymphocytes, and regulating the differentiation of T helper cells.VDR Measuring transcriptional activity of vitamin D receptor. VDR is amember of the steroid receptor superfamily. In its ligand bound state,VDR forms heterodimers with RXR and regulates gene expression by bindingto specific hormone response elements. The VDR-RXR heterodimer has beenshown to bind to VD-responsive elements (VDRE) of osteocalcin andosteopontin genes to stimulate transcription of these genes. YY1Measuring transcriptional activity of YY1. YY1 is a ubiquitouslydistributed transcription factor belonging to the GLI-Kruppel class ofzinc finger proteins. The protein is involved in repressing andactivating a diverse number of promoters. YY1 may direct histonedeacetylases and histone acetyltransferases to a promoter in order toactivate or repress the promoter, thus implicating histone modificationin the function of YY1.

In one embodiment said response element of the target antigen is anuclear response element in the nucleus of the cell. In anotherembodiment said response element is located on a plasmid in the cell. Inone embodiment the assay comprises the preliminary step of transfectionof the cells with an expression vector comprising the DNA sequencecoding for the reporter gene under the control of the target antigenresponse element. In one embodiment the target antigen is a cell surfacereceptor.

In one embodiment the binding to the target antigen and thefunctionality of the antibody or ligand are measured in the same vial orwell. In one embodiment the binding to the target antigen and thefunctionality of the antibody or ligand are measured in the same testingmedium. The advantage of the new assay described herein that no washingsteps are required. Preferably the testing medium is a medium thatprovides conditions for cells to be viable for up to 48 hours. Suitablemedia are for example Fluorobright or DMEM, as outlined in the examples.In one embodiment the assay is performed in a microtiterplate. In oneembodiment the microtiterplate is suitable for high throughputscreening. The assay of the present invention can be performed in anyformat that allows for rapid preparation, processing, and analysis ofmultiple reactions. This can be, for example, in multi-well assay plates(e.g., 96 wells or 386 wells). Stock solutions for various agents can bemade manually or robotically, and all subsequent pipetting, diluting,mixing, distribution, washing, incubating, sample readout, datacollection and analysis can be done robotically using commerciallyavailable analysis software, robotics, and detection instrumentationcapable of detecting FRET, BRET or AlphaScreen signals.

In one embodiment the steps iii) and iv) are performed consecutively orsimultaneously.

In one embodiment the FRET is time resolved FRET. In one embodiment theFRET energy donor compound is Terbium cryptate and/or the FRET energyacceptor compound is d2. In one embodiment the target antigen and theresponse element are part of the NF-κB pathway. In one embodiment thetarget antigen is selected from a target antigen on Toll-like receptors,TNF receptors, T cell receptor and B cell receptor. Non-limitingexamples of antibodies that upon binding to its target result inmodulation of the activity of NF-κB are anti-CD40 antibodies, anti-DR5antibodies, anti-DR4 antibodies, anti-41BB antibodies, anti-Ox40antibodies and anti-GITR antibodies.

In one embodiment the response element is a NF-κB response element. Inone embodiment said response element comprises one or more of thefollowing DNA repeats GGGAATTTCC (SEQ ID NO: 1), GGGGACTTTCC (SEQ IDNO:2), GGGACTTTCC (SEQ ID NO:3), GGGACTTCC (SEQ ID NO:4), ATTGTAGCGTA(SEQ ID NO: 5). In one embodiment said response element comprises 3 to6, 3 or 6 of the DNA repeats mentioned above. In one embodiment saidresponse element comprises 3 to 6, 3 or 6 of the DNA repeats mentionedabove and 1, 2, 3 or 4 additional nucleotides.

In one embodiment said response element comprises a DNA sequence of

(SEQ ID NO: 6) GGGAATTT CCGGGGACTT TCCGGGAATTTCCGGGGACT TTCCGGGAAT TTCC,(SEQ ID NO: 7) GGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCC, (SEQ ID NO: 8)GGGACTTCCGGGACTTTCCGGGACTTTCCGGGACTTTCCGGGACTTTCCG GGACTTTCC,(SEQ ID NO: 9) GGGACTTTCCATTGTAGCGTAGGGACTTTCCATTGTAGCGTAGGGCTTTCCATTGTAGCGTAGGGCTTTCC,

In one embodiment the binding and/or functionality of the antibody orligand is compared to the binding and/or functionality of a benchmarkantibody or ligand (both measured with the assay as described herein). Abenchmark antibody or ligand is a prior art antibody or ligand thatbinds to the same antigen as the antibody or ligand to be tested and hasknown properties (e.g. binding and/or functionality).

In one embodiment the assay comprises the preliminary step oftransfection of the cells with an expression vector comprising the DNAsequence encoding for the reporter gene under the control of the targetantigen response element.

In one embodiment 1000 to 40 000 cells per well are provided in step i).In a preferred embodiment 5000 to 30000 cells or 5000 to 10000 cells perwell are provided in step i).

In one embodiment the antibody or ligand is provided in step ii) toachieve a concentration of 120-0.02 nM antibody or ligand per well.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

For combining functionality and binding assessment, two assays werefirst optimized and then combined: The luciferase assay which providesinformation about the functionality of an antibody and an assay whichallows studying protein-protein interactions such as the FRET assay. Fora combination of both assays, reporter cell lines transfected with thetarget antigen named “Receptor X” fused to a SNAP tag and labeled with adonor fluorophore are needed. Prior to transfection, the reporter celllines HeLa NFκB-Luc and HEK NFκB-Luc-GFP were evaluated for their NFκBactivation by performing a luciferase assay. Instead of anti-Receptor Xantibodies, TNFα was used to test the activation of the NFκB pathway,resulting in an expression of luciferase.

TABLE 1 List of Abbreviations Abbreviation Meaning AGTO⁶-alkylguanine-DNA alkyltransferase Alpha Amplified LuminescentProximity Homogeneous Assay AMP Adenosine monophosphate APCAllophycocyanin ATP Adenosine triphosphate BG O⁶-benzylguanin CO₂ Carbondioxide CoA Coenzyme A DMEM Dulbeccos Modified Eagle Medium DMSODimethyl sulfoxide FACS Fluorescence-activated cell sorting FBS Fetalbovine serum FRET Fluorescence Resonance Energy Transfer GFP GreenFluorescent Protein GlcNAc N-acetyl glucosamine HEK Human EmbryonicKidney HeLa Henrietta Lacks HTRF Homogeneous Time Resolved Resonance HTSHigh Throughput Screening hu Human IKK IκB kinase IκB Inhibitory kappa Bprotein kDa Kilodalton Luc Luciferase Lumi4-Tb Terbium cryptate minPMinimal promoter MW Molecular weight NFκB Nuclear Factor-kappa B PBSPhosphate buffered saline PP_(i) Pyrophosphate RE Response element RLURelative Luminescence Units RT Room temperature SPR Surface PlasmonResonance TNF Tumor Necrosis Factor TR Time Resolved WGA Wheat GermAgglutinin

TABLE 2 Materials Material Tradename Manufacturer ReagentsLipofectamine ® 2000 Invitrogen, USA Lipofectamine ® LTX Invitrogen, USAX-tremeGene HP Roche, Switzerland Dimethyl sulfoxide Sigma-Aldrich, USADulbeccos Modified Eagle Gibco, USA Medium Fetal bovine serum Gibco, USASNAP-Lumi4-Tb Cisbio, France Tag-lite ® reaction buffer 5x Cisbio,France Cell Dissociation Buffer Gibco, USA WGA-Terbium (0.1 mg/ml)Cisbio, France ONE-Glo ™ Reagent Promega, USA Luciferin 1000 ReagentPromega, USA Opti-MEM ®I Reduced Gibco, USA Serum Medium FluoroBrite ™DMEM Gibco, USA Phosphate Buffered Saline Gibco, USA GlutaMax-I Gibco,USA Hygromycin B Roche, Switzerland Devices Tecan Infinite ® M1000 ProTecan, Austria Vi-cell ™ XR Beckman Coulter, USA Viktor³ ™ 1420Multilabel PerkinElmer, USA Counter SpectraMax M5/M5e plate readerMolecular Devices, USA Antibodies Antibody-I Roche, SwitzerlandAntibody-II Roche, Switzerland Antibody-III Roche, SwitzerlandAntibody-IV Roche, Switzerland Antibody-V Roche, SwitzerlandAnti-human-IgG-d2 Cisbio, France anti-hu IgG Fcγ-specific Jackson, USAgoat IgG F(ab)2

Example 1: Transient Transfection and Labeling of Cells withSNAP-Receptor X Fusion

Optimization of the Transient Transfection of HeLa NFκB-Luc Cells

The HeLa NFκB-Luc cells had to be transfected with a plasmid carryingthe gene encoding for a SNAP-tag® Receptor X fusion. To find out thebest transfection method, the cell number seeded per well as well asthree different transfection reagents (Lipofectamine® 2000,Lipofectamine® LTX and X-tremeGene HP DNA transfection reagent) wereevaluated.

Evaluation of Cell Number

Six different cell numbers were seeded in a 6-well-plate (300 000, 400000 up to 800 000 viable cells per well) to find the optimal number tobe used to achieve 70% confluency after an incubation time of 24 h.

Evaluation of Transfection Reagents

For the evaluation of the transfection reagent, 500 000 viable HeLaNFκB-Luc cells per well were seeded in a 6 well plate and incubated for24 h at 37° C., 5% CO2. Cells were washed with 1 ml PBS, 2 ml ofpre-warmed growth medium added and each well then treated with adifferent transfection reagent.

The first transfection mix was prepared by mixing 150 μl of Opti-MEM®IReduced Serum Medium and 10 μl of Lipofectamine® 2000 DNA transfectionreagent in a sterile 1.5 ml Eppendorf reaction tube. In a second tube150 μl Opti-MEM®I Reduced Serum Medium was mixed with 3.5 μg of theSNAP-Receptor X plasmid. After an incubation time of at least 5 minutesat room temperature, the content of both tubes was mixed and incubatedfor 25 minutes at room temperature.

For the second transfection mix, 1.25 μg of the plasmid was diluted in500 μl of Opti-MEM®I Reduced Serum Medium. Subsequently, 5 μl ofLipofectamine® LTX was added, mixed gently and incubated for 25 minutesat room temperature.

The third transfection mix was prepared by diluting 2 μg of the plasmidin 200 μl of Opti-MEM®I Reduced Serum Medium. Then, 6 μl of X-tremeGENEHP DNA transfection reagent was added and incubated for 30 minutes atroom temperature.

Each transfection mixture was added to the cells in a drop wise manner.The 6-well-plate was gently swirled to ensure even distribution over theentire well. The cells were then incubated for 24 h in a humidifiedincubator at 37° C. and 5% CO2.

After the incubation, the medium with the transfection mixture wascarefully removed and the transfected cells were washed once with 3 mlof PBS. For labeling of the cells with SNAP-Lumi4-Tb, 1.5 ml dilutedlabeling reagent (100 nM in Tag-lite® buffer) was added to thetransfected cells and incubated for 1 h in a humidified incubator.Afterwards, the labeling reagent was removed and the cells were detachedby using 500 μl Cell Dissociation Buffer. The detached cells wereresuspended in 2.5 ml of PBS, transferred to a 15 ml Falcon tube andwashed three times with 5 ml of phosphate buffered saline (PBS) by a 5min centrifugation step at 300 g. The cell pellet was resuspended in 140μl Tag-lite® reaction buffer. 100 μl from the cell suspension was usedfor a 1:5 dilution to determine the cell viability and the viable cellconcentration by the Vi-cell™ XR from Beckmann Coulter. To evaluate theprotein expression after transfection, the remaining 40 μl of cellsuspension were distributed in 2 wells of a white 384-well-plate withflat bottom to measure the terbium signal at a wavelength of 615 nm in aplate reader (Victor3™). In order to compare the level of transfectionbetween the different transfection methods, the terbium signal wasnormalized to a cell number of 10 000 viable cells per well.

Results

In order to find out the best transfection method, three differenttransfection reagents were tested in a 6-well-plate: Lipofectamine 2000,Lipofectamine LTX and Xtreme Gene HP. For each reagent, 500 000 viablecells per well were seeded out. The SNAP tag fusion protein wasafterwards labeled with the donor fluorophore terbium to performprospective Tag-lite® experiments. Additionally, the terbium labelallows determination of the transfection efficiency when measuringfluorescence at a wavelength of 615 nm. Moreover, the viability of thecells after transfection and labeling was determined (FIG. 1 ). FIG. 1shows that Lipofectamine LTX was the best reagent for transfecting HeLacells. Besides the best viability of the cells after transfection andlabeling, also the highest terbium signal could be achieved. Incontrast, Xtreme Gene HP as well as Lipofectamine 2000 killed more cellsand the level of transfection was lower.

Upscaling of the Transfection from 6-Well Plate to T150 Cell CultureFlasks

After determining the best transfection method a larger number of cellshad to be transfected and labeled for the planned experiments for theongoing project. In addition to HeLa NFκB-Luc cells, also HEKNFκB-Luc-GFP cells had to be transfected. Therefore, 15 Mio viable HeLaNFκB-Luc cells and 11 Mio viable HEK NFκB-Luc-GFP cells were seeded outone day prior to transfection in a T150 cell culture flask in 25 ml ofDMEM+10% FBS. The transfection complex for the HeLa cells was preparedby adding 18.75 μg of the SNAP-Receptor X plasmid to 7.5 ml ofOpti-MEM®I Reduced Serum Medium, whereas for the HEK cells 37.5 μg ofthe DNA was diluted in the same volume of transfection medium. Aftergently mixing the dilution, 75 μl of Lipofectamine® LTX DNA transfectionreagent was added. After washing the cells in the T150 cell cultureflask, the transfection complex was added drop wise and incubated 24 hin a humidified incubator. Afterwards, the transfected cells were washedagain and labeled with 10 ml of a 100 nM labeling reagent dilution byincubation at 37° C. for 1 h in a humidified incubator. The cells werewashed three times in PBS and counted using the Beckmann CoulterVi-cell™ XR. The cells were then centrifuged for 5 minutes at 300 g andresuspended in Tag-lite® reaction buffer to determine the proteinexpression. Therefore, 10 000 viable cells per well were used in avolume of 20 μl Tag-lite® reaction buffer. The terbium signal wasmeasured in duplicates at a wavelength of 615 nm. The cell suspensionwas again centrifuged, the supernatant discarded and the pelletresuspended in an appropriate volume of freezing medium which was 90%heat inactivated FBS and 10% DMSO to get 1 Mio cells/ml. Aliquots of 0.5and 1 ml were frozen at −80° C. in cryovials with a Nalgene® cryogenicvial freezing box containing Isopropanol.

Transient transfection of stable Receptor X expressing cells withReceptor X-SNAP were carried out in the same way as described above butusing 4 Mio cells per T75 flasks and 10 μg DNA diluted in 4 ml OptiMEM®I Reduced Serum Medium and 37.5 μl Lipofectamine® LTX. These cellsare termed “supertransfected” therein.

Results

For the ongoing project, a larger amount of cells was needed. Thus, HEKNFκB-Luc-GFP and HeLa NFκB-Luc cells were transfected in a T150 cellculture flask with Lipofectamine LTX and the terbium signal wasdetermined (FIG. 2 ).

The terbium signals as well as the viabilities show a similar result forboth cell lines. Thus, it was assumed that the expression ofSNAP-Receptor X fusion protein on the cells surface was similar andtherefore, the results of prospective assays with these cell lines couldbe compared.

Example 2: Indirect Binding Assay by Tag-Lite®

General Tag-Lite® Protocol

A two-fold dilution series of an anti-human-Receptor X antibody calledAntibody-I in Tag-lite® reaction buffer was prepared ranging from 100 nMto 0.2 nM. The anti-human-IgG-d2 detection antibody was diluted in thesame buffer to a final concentration of 150 nM per well. HEKNFκB-Luc-GFP cells transiently transfected with the SNAP-Receptor Xplasmid and labeled with terbium were used in each assay. The cell linewas thawed and washed with 10 ml of PBS by centrifugation for 5 minutesat 350 g. The supernatant was discarded and the pellet was resuspendedin Tag-lite® reaction buffer to obtain 1 Mio cells/ml. In a Tag-lite®assay, the cells are not used at a certain cell number, but instead theyare adjusted to a certain Tb signal. To find out how many cells have tobe used per well in the assay, 10 μl of the resuspended cells (10000cells per well) were mixed with 10 μl of Tag-lite® reaction buffer in a384-well-plate in triplicates and the terbium signal at 615 nmdetermined in a Victor3™ plate reader. For the Tag-lite® assay the cellnumber was then adjusted to obtain a terbium signal between 20 000 and30 000 counts per well determined by the Victor3™ plate reader. TheTag-lite® assay was pipetted using 10 μl of the diluted cells, 5 μl ofanti-human-IgG-d2 and 5 μl of the dilution series of the antibody. As ablank, 10 μl of the cells, 5 μl of the secondary antibody and 5 μl ofbuffer was pipetted. All measurements were done in triplicates. Theplate was measured after 0 h, 2 h and 4 h of incubation at roomtemperature with the Tecan Infinite® M1000 Pro plate reader.

All following Tag-lite® experiments including the comparison ofdifferent anti-Receptor X IgGs and constructs, evaluation of variousdetection antibody concentrations, incubation temperature and theinfluence of incubation media were pipetted using this protocol.Exceptions or changes from the general protocol are stated in the resultsection.

Results

After transfection and labeling, both cell lines were tested in anindirect Tag-lite® binding assay with two different IgGs (Antibody-I and-II) targeting Receptor X (FIG. 3 ). FIG. 3 shows that Antibody-I bindsbetter to both cell lines (Receptor X expressing HEK NFκB-Luc-GFP andHeLa NFκB-Luc cells) than Antibody-II. The binding signal of Antibody-Iwas for both cell lines of the same strength. For Antibody-II only aweak signal can be detected for the HeLa NFκB-Luc-Receptor X-Tb cellsand no binding can be seen at HEK NFκB-Luc-GFP-Receptor X-Tb cells.Thus, Antibody-I was used for the ongoing experiments.

The d2 labeled anti-human-IgG which was used as secondary antibodyshould be at least in a threefold molar excess compared to the primaryantibody. Instead of using a concentration of 150 nM as in the previousassays, 75 nM final per well were tested and the results compared (FIG.4 ). FIG. 4 shows that there was no significant difference betweenconcentrations of 150 or 75 nM of secondary antibody. For the bindingcurve with 150 nM, a KD value of 0.19 nM±0.05 nM (R2=0.93) wasdetermined, whereas for the concentration of 75 nM a KD of 0.16 nM±0.03nM (R2=0.95) was found. Thus, if using the primary antibody inconcentrations up to 20 nM, 75 nM for the secondary antibody was used inall ongoing experiments.

Instead of using Antibody-I which is an IgG, also two different ReceptorX antibody constructs were tested for their binding, namely Antibody-IIIand -IV (FIG. 5 ).

All antibodies and antibody like constructs show similar binding and KDvalues with 0.11 nM±0.03 nM (R2=0.85) for Antibody I, 0.18 nM±0.02 nM(R2=0.97) for Antibody-III and 0.28 nM±0.04 nM (R2=0.98) forAntibody-IV.

For the combination of a luciferase assay with the Tag-lite® bindingassay it might be necessary to incubate the cells at 37° C. rather thanroom temperature (RT) which is commonly used for the binding assay.Therefore, binding at 37° C. had to be assessed (FIG. 6 ). The KD valuedetermined for the incubation at RT was with 0.18 nM±0.02 nM (R2=0.97)only slightly lower than for the incubation at 37° C., (KD was 0.43nM±0.18 nM (R2=0.85)). However, binding for both was in the lownanomolar range and therefore comparably strong.

Instead of using Tag-lite® buffer or PBS for the binding assay, othermedia were tested which might be more suitable for the luciferase assay(FIG. 7 ). FIG. 7 shows that PBS and DMEM decreased the binding of theantibody construct to the receptor. DMEM/10% FBS or FluoroBrite DMEM/10%FBS showed similar results compared to the generally used Tag-lite®buffer and are therefore suitable alternatives for the combinationassay.

Time Dependent Stability of the Receptor X on Transiently TransfectedCells after Thawing

After transient transfection of the HEK NFκB-Luc-GFP cells with theSNAP-Receptor X plasmid, the cells were frozen and used for the dailyexperiments. To determine the stability of the receptors after takingthawed transiently transfected cells in culture, an indirect Tag-lite®binding assay was performed every day for three days. Therefore, thawedcells were splitted into 4 wells of a 6-well-plate and cultured in 4 mlFluoroBrite™ DMEM+10% FBS. Every day, the cells of one well of the6-well-plate were detached, 5 minutes centrifuged at 350 g andresuspended in FluoroBrite™ DMEM+10% FBS to a final concentration of 1Mio cells/ml. 10 000 cells per well were mixed then with a two-folddilution series of Antibody-III in FluoroBrite™ DMEM+10% FBS mediumranging from 80 nM to 0.16 nM to test the binding. The anti-human-IgG-d2detection antibody was diluted in the same medium to a concentration of75 nM final per well. The terbium signal was monitored daily.

Results

The presence of the receptor on the cell surface of the transientlytransfected cells was monitored over time. Directly after thawing thecells (day 0) and also the following 3 days, an indirect Tag-lite®binding assay (FIG. 8 ) as well as a measurement of the terbium signalof the cells (FIG. 9 ) was performed by using 10 000 thawed or culturedcells per well. FIG. 8 shows that the binding signal significantlydecreased from day 0 to day 1 and was completely absent at day 2 and 3.The ratio of 665 to 620 nm*10 000 was for time point day 0 wasapproximately 10 000, whereas 1 day later, Bmax was approximately 3000,which represents 70% of the original signal. The KD value for time pointday 0 and day 1 were both in the nanomolar range with 0.49 nM±0.08 nM(R2=0.98), and 0.12 nM±0.03 nM (R2=0.87) respectively.

FIG. 9 is in line with the results depicted in FIG. 8 showing a decreasein terbium signal over time. In contrast, the viability increased fromday to day.

After in depth evaluation of the parameters for the Tag-lite® assay alsothe conditions for the luciferase assay had to be optimized.

Evaluation of the HTRF Raw Data

For the analysis of the assays, the raw data were first edited byMicrosoft Excel. In a HTRF assay variations in the results can occurfrom well to well due to the pipetting steps of the cells, mediumadditives and from the number of lysed cells per well. To minimize thosevariations, the emission of the acceptor was normalized to the emissionof the donor signal in each well by calculating the ratio of 665 nm to620 nm:“ratio=” “665 nm”/“620 nm” “*10000”.

The calculated ratio values were evaluated by the software calledGraphPad Prism 6.0. The binding curves were fitted with nonlinearregression. Bmax and KD were determined using the “One site—specificbinding” model using the equation:“Y=”(“B”_“max” “* X”)/“(” “K”_“D” “+X)”).

Example 3: Luciferase Assay

Evaluation of the Luciferase Activation of Two Different Cell Lines

HeLa NFκB-Luc cells used for transient transfection as well as alreadystably transfected NFκB-Luc-Receptor X cells were compared for theirluciferase activity upon stimulation with TNFα

The method used was the Luciferase 1000 assay system (Promega).Therefore, both cell lines were seeded in a white 96-well-plate with acell number of 20 000 cells per well in 100 μl growth medium (DMEM+10%FBS+1% GlutaMax-I+200 μg/ml Hygromycin B) the day before activation. Inaddition, the same cells were also seeded in 3 wells of a transparent96-well-plate to determine the confluency, attachment and contaminationstatus microscopically before activation. After 24 h of incubation at37° C., 5% CO2, a two-fold dilution series of TNFα was prepared rangingfrom 25 ng/ml to 0.8 ng/ml. 100 μl of each dilution was added to thecells in triplicates followed by an incubation step for 6 h at 37° C.and 5% CO2. Afterwards, the cells were washed three times with 200 μlPBS per well by a 5 minute centrifugation step at 350 g. For detectionof the produced luciferase, the cells had to be lysed. Therefore, 40 μlof lysis buffer was added to each well and incubated for 2 hours at −80°C. to ensure lysis. After adjusting the cells to room temperature, 100μl of Luciferase 1000 assay reagent was added to each well in the darkand light emission at all wavelengths of the entire visible spectrum wasmeasured immediately by the SpectraMax M5/M5e plate reader with 500 msintegration time. As a blank, the signal of the lysed cells with theluciferase reagent was subtracted.

The light which was produced as a by-product of the reaction wascollected by the luminometer from the entire visible spectrum.

The same protocol was also used for the comparison of the Hela NFκB-Luccells with the HEK NFκB-Luc-GFP cell line.

Different Activation Methods in Comparison

The same assay as described above was performed in triplicates, but onlywith transiently transfected HEK NFκB-Luc-GFP-SNAP-Receptor X cellsseeded at 80 000 cells per well in DMEM+10% FBS. Besides the activationthrough TNFα receptors, Antibody-I was used to bind to Receptor X whichshould also be able to activate the NFκB pathway upon oligomerisation.Therefore, a four-fold dilution series of Antibody-I ranging from 120 to0.03 nM was prepared and 50 μl of each concentration added to the cellsfollowed by an incubation step for 15 minutes at 37° C. and 5% CO2. Forhypercrosslinking of the receptor which is required to activate the NFκBpathway, 50 μl of a secondary antibody (anti-hu IgG Fcγ-specific goatIgG F(ab)2) was used. The concentration of this secondary antibody waskept constant at 480 nM which translates into an at least four-foldmolar excess compared to the primary antibody.

Comparison of the Luciferase 1000 and the ONE-Glo™ Luciferase AssaySystem

Besides the Luciferase 1000 assay system, also the ONE-Glo™ Luciferaseassay system was tested and the results compared. Therefore, transientlytransfected HEK NFκB-Luc-GFP-SNAP-Receptor X cells were tested incomparison to stably transfected HEK NFκB-Luc-GFP-Receptor X cells whichserved as a positive control. Additionally, the activation by TNFα aswell as by Receptor X was analyzed.

Both cell lines were seeded in two white 96-well-plates (one plate foreach assay system) with a cell number of 30 000 cells per well in 150 μlDMEM+10% FBS the day before activation. In addition, the same cells werealso seeded in 3 wells of a transparent 96-well-plate to determine theconfluency, attachment and contamination status microscopically beforeactivation. After 24 h incubation at 37° C. and 5% CO2, 10 nM of theprimary Antibody-III was added to the wells tested for the Receptor Xactivation. After another 15 minutes of incubation, 40 nM of thesecondary antibody or 50 ng/ml TNFα was added. The plates were incubatedfor 48 h at 37° C. and 5% CO2.

One plate was treated as described above for the Luciferase 1000 assaysystem. Briefly, the cells were washed, lysed, frozen, thawed andmeasured after adding the luciferase reagent. As a blank, the signal ofthe lysed cells without activation was subtracted. The other plate wasused for the ONE-Glo™ Luciferase assay system. Therefore, an appropriatevolume of the supernatant of each well was removed to have a finalvolume of 100 μl per well. 100 μl of ONE-Glo™ Luciferase reagent wasadded per well. After 5 minutes of incubation in the dark, lightemission was measured by the SpectraMax M5/M5e plate reader with 500 msintegration time. As a blank, the signal of the lysed non-activatedcells with the luciferase reagent was subtracted. The measurement wasdone in triplicates.

Results

HeLa NFκB-Luc cells were first compared in a luciferase assay with HeLaNFκB Luc-Receptor X cells. The latter are known for their ability toactivate the NFκB pathway by TNFα receptors, thus they were used as apositive control (data not shown). Almost the same luminescence signalwas reached by the HeLa NFκB-Luc cells as by the positive control. Thus,the NFκB pathway could be activated well by TNFα meaning that the cellscould be used for transfection of the plasmid with the genes encodingfor SNAP-Receptor X.

HEK NFκB-Luc-GFP cells were tested and compared to HeLa NFκB-Luc cells(data not shown). This cell line would preferably be used to develop thecombinatory assay due to its second reporter gene which enables anadditional tool for detecting gene expression.

HEK NFκB-Luc-GFP cells showed a higher signal than HeLa NFκB-Luc cells.As both cell lines can be used for a luciferase assay, each of them wastransfected with the SNAP-Receptor X plasmid.

After transfection and labeling of the HEK NFκB-Luc-GFP cells withReceptor X-SNAP, Antibody-I was used for the initial luciferase assayand compared to the activation by TNFα. Antibody-I as the primaryantibody as well as the secondary antibody were used alone as negativecontrols (FIG. 10 ). FIG. 10 shows a strong activation of the NFκBpathway by TNFα. Antibody-I and the secondary antibody alone were notable to activate the pathway. There was only a very weak luminescencesignal detected for the combination of both antibodies. In conclusion,Antibody-I was determined to be the best binder, but did not show goodfunctionality in this setting.

Furthermore, two different luciferase assay systems were evaluated, theONE-Glo™ and the Luciferase 1000 system (FIG. 11 ). Therefore, TNFα wasused for activation of the NFκB pathway of stably and transientlytransfected Receptor X expressing HEK NFκB-Luc-GFP cells. Instead of a 6h incubation time after activation, 48 h were used to give the cellsmore time for expressing luciferase. FIG. 11 shows that the ONE-Glo™Luciferase assay system was more sensitive to the light released by theluciferase catalyzed reaction from luciferin to oxyluciferin. Due to thehigher luminescence signal as well as the faster and easier to performassay procedure, the ONE-Glo™ Luciferase assay system was used for theongoing experiments.

In the following experiment the incubation times of 6 h and 48 h werecompared by using the same cells as in the previous experiment.Antibody-III as the primary antibody was used at a concentration of 10nM and the secondary antibody (anti-hu IgG Fcγ-specific goat IgG F(ab)2)was used at a concentration of 40 nM (FIG. 12 ).

The stably transfected HEK NFκB-Luc-GFP-Receptor X cells clearlyindicated that after 48 h of incubation, more luciferase was produced bythe cells, resulting in a stronger bioluminescent signal. Antibody-IIIalone activated to a lower extent the pathway which was expected as itcan already trimerise the receptor. The signals of the transientlytransfected cells were significantly lower. Especially for the 6 hincubation there was no major difference in bioluminescence between thecombination of both antibodies and each antibody on its own whereasthere was a slight difference for the 48 h value.

“Down-Scaling” from 96- to 384-Well-Plates

For the combination assay of functionality and binding, it was necessaryto transfer the luciferase assay which is usually done in a96-well-plate to a 384-well-plate. The ONE-Glo™ Luciferase assay wasperformed for a 96-well-plate, but instead of using 30 000 cells perwell, only 5000 cells were used. The primary and secondary antibodieswere kept at a constant concentration of 10 nM and 40 nM, respectively,whereas TNFα was used at a concentration of 50 ng/ml. After 24 h ofincubation at 37° C. and 5% CO2, an appropriate volume of thesupernatant was removed to have a volume of 15 μl remaining in eachwell. 15 μl of ONE-Glo™ Luciferase reagent was added per well. Themeasurement was done with stable HEK NFκB-Luc-GFP-Receptor X cells intriplicates.

Additionally two different cell numbers (5 000 and 10 000 cells perwell) were evaluated using the same protocol as described above.

Results

Combining functionality and binding assessment was thought to be usedfor High-Throughput-Screenings (HTS). For this reason and also for thereason of costs and to bring the luciferase assay in line with Tag-lite®experiments, down-scaling from a 96-well-plate to 384 wells was tested(FIG. 13 ).

In overall, FIG. 13 shows a slightly stronger signal for the 96 than forthe 384-well-plate. Nevertheless, even though the signal was lower in384 wells than in 96 wells, the result regarding the activation orfunctionality remains the same. Thus, the ongoing luciferase assays wereperformed in 384-well-plates.

For the down-scaling of the luciferase assay, a cell number of 5 000viable cells per well was calculated, but for the binding experimentssometimes 10 000 cells are needed per well to have a high enoughfluorescent signal of the donor fluorophore for the FRET signal. Thefeasibility of using either 5 000 or 10 000 was tested with the ONE-Glo™Luciferase assay system using stably transfected HEKNFκB-Luc-GFP-Receptor X cells (FIG. 14 ). FIG. 14 shows that there waseven a stronger signal for the bioluminescence when using 10 000 viablecells per well. Despite keeping the other parameters—such as the amountof added antibodies—constant, more luciferase could be produced by thecells resulting in a stronger bioluminescent signal.

Now, all the parameters for a combination of a binding and a luciferaseassay were aligned. Unfortunately, neither of the cell line was suitablefor a combination assay. There were stably transfected HEKNFκB-Luc-GFP-Receptor X cells available, which show good results in afunctionality assay, but cannot be used for a normal Tag-lite® assay dueto the lack of the donor fluorophore and there were transientlytransfected HEK NFκB-Luc-GFP-Receptor X-Tb cells available, which showgood results in the binding assay, but cannot be activated very well.Therefore, two approaches were tested. On one hand a WGA based FRETassay on stably transfected cells and on the other hand asupertransfection of the stably transfected cells with the ReceptorX-SNAP construct.

Optimization of the Concentration of the Primary Antibody

All the luciferase assays were performed using 10 nM of the primaryantibody and 40 nM of the secondary. The aim of this assay was totitrate the first antibody with five dilutions and to keep the secondaryantibody in a 1:4 ratio for detection.

The ONE-Glo™ Luciferase assay was performed using transientlytransfected HEK NFκB-Luc-GFP SNAP-Receptor X cells, stably transfectedHEK NFκB-Luc-GFP-Receptor X cells as well as the transientlysupertransfected cells. For all three cell lines 5 000 cells per wellwere seeded out in a 384-well-plate in 15 μl DMEM+10% FBS and incubatedfor at least 12 h at 37° C. and 5% CO2 before activation. A two-folddilution series of the primary Antibody-III was prepared and added tothe wells ranging from 40 nM to 2.5 nM per well. After 15 minutes ofincubation, the secondary antibody (anti-hu IgG Fcγ-specific goat IgGF(ab)2) was added to the wells at concentrations ranging from 160 nM to10 nM per well, to keep the secondary antibody in a fourfold molarexcess compared to the primary antibody. The plates were incubated at37° C. and 5% CO2. After 24 h of incubation, an appropriate volume ofthe supernatant was removed to have a final volume of 15 μl per wellremaining. 15 μl of ONE-Glo™ Luciferase reagent was added per well.After 5 minutes of incubation in the dark, light emission was measuredby the SpectraMax M5/M5e plate reader with 500 ms integration time. As ablank, the signal of the lysed non-activated cells with the luciferasereagent was subtracted. The measurement was done in triplicates.

Ratio Optimization of the Cross-Linking Antibody

For the functionality assay, the secondary antibody (anti-hu IgGFcγ-specific goat IgG F(ab)2) was used for cross-linking. To combine theluciferase assay with the binding assay, an antibody was necessary whichis labeled with the acceptor fluorophore d2. Either the anti-hu IgGFcγ-specific goat IgG F(ab)2 had to be labeled or the anti-human-IgG-d2from CisBio could be used. The primary antibody was kept at a constantconcentration of 40 nM, which was optimized in the assay before, whereasthe ratio of primary antibody to secondary antibody changed from 1:1 to1:5 ratio.

Example 4: Indirect WGA-HTRF

The anti-human-IgG-d2 detection antibody was diluted in Tag-lite®reaction buffer to a concentration of 75 nM final per well. A two-folddilution series of Antibody-III in Tag-lite® reaction buffer wasprepared ranging from 1.56 nM to 0.01 nM. Stable HEK NFκB-Luc-GFPReceptor X cells were thawed and washed with 10 ml of PBS bycentrifugation for 5 minutes at 350 g. The supernatant was discarded andthe pellet was resuspended in Tag-lite® reaction buffer to have 1 Miocells/ml. 0.05 ng/μl WGA-Terbium was added to the cell suspension andincubated at room temperature for 30 minutes. Afterwards the cells werewashed again and resuspended in an appropriate volume of Tag-lite®reaction buffer to have a final concentration of 1 Mio cells/ml.

10 μl of the resuspended WGA-Terbium labeled cells were mixed with 10 μlof Tag-lite® reaction buffer in a 384-well-plate. The terbium signal wasdetermined in triplicates at a wavelength of 615 nm using the Victor3™plate reader. The assay was pipetted using 10 μl of cells with a certainterbium signal, 5 μl of anti-human-IgG-d2 and for the titration 5 μl ofa dilution of the antibody. As a blank, 10 μl of the cells, 5 μl of thesecondary antibody and 5 μl of buffer was pipetted. The assay waspipetted in triplicates in a 384-well-plate and measured after 0 h, 2 hand 4 h of incubation at room temperature with the Tecan Infinite® M1000Pro plate reader. The evaluation of the raw data was done as describedabove.

Results

The stably transfected cells were incubated with the terbium-conjugatedlectin WGA and an indirect HTRF assay was set-up by usinganti-human-IgG-d2 as an acceptor labeled secondary antibody (FIG. 15 ).

In FIG. 15 a binding signal of Antibody-III was detected compared to thenegative control. The measured values were fitted equally with theTag-lite® experiments, so that the KD value can be determined andcompared. The KD value in this experiment was 0.15 nM±0.03 nM (R2=0.97).

Example 5: Combination Assay

For the combination assay, a two-fold dilution series of Antibody-III inFluoroBrite DMEM+10% FBS was prepared ranging from 200 nM to 0.1 nM.This leads to concentrations ranging from 40 nM to 0.02 nM final perwell. The anti-human-IgG-d2 detection antibody was diluted in the samemedium to a final concentration of 120 nM per well. HEK NFκB-Luc-GFPcells which were transiently supertransfected with the SNAP-Receptor Xplasmid and labeled with terbium were used. The cell line was thawed andwashed with 10 ml of PBS by centrifugation for 5 minutes at 350 g. Thesupernatant was discarded and the pellet was resuspended in FluoroBriteDMEM+10% FBS. The assay was pipetted in a 384-well-plate using 10 000cells in 15 μl medium, 5 μl of anti-human-IgG-d2 and 5 μl of thedilution series of the antibody per well. As a blank, 15 μl of thecells, 5 μl of the secondary antibody and 5 μl of buffer was pipetted.All measurements were done in triplicates. The plate was measured after0 h, 2 h and 4 h incubation at 37° C. and 5% CO2 with the TecanInfinite® M1000 Pro plate reader. After 24 h of incubation, 10 μl ofsupernatant was removed to have a final volume of 15 μl per wellremaining. 15 μl of ONE-Glo™ Luciferase reagent was added per well.After 5 minutes of incubation in the dark, light emission was measuredby the SpectraMax M5/M5e plate reader with 500 ms integration time. As ablank, the signal of the lysed non-activated cells with the luciferasereagent was subtracted.

Results

Evaluation of a Cell Line Suitable for Combining Functionality andBinding Assessment

A new approach is to “transiently supertransfect” stably transfected HEKNFκB-Luc-GFP-Receptor X cells. This means the plasmid encoding forSNAP-Receptor X was used to transfect the HEK NFκB-Luc-GFP-Receptor Xcells again with Receptor X fused to the SNAP-tag®. Afterwards, thecells were labeled with terbium and could be used for a normal indirectTag-lite® binding experiment (FIG. 16 ). The transiently transfectedcells were used as a control as they showed good binding withAntibody-III in the initial experiments. FIG. 16 shows that there was alower ratio for the transiently supertransfected cells compared to thetransiently transfected cells. However, the signal was high enough toobtain a nice curve fit and determine the KD value. The KD was the samefor both cell types with 0.09 nM±0.02 nM (R2=0.92) was and also 0.09nM±0.02 nM (R2=0.96) for the HEK NFκB-Luc-GFPReceptor X-Tbsupertransfected and transfected cells, respectively.

Thus, those supertransfected cells have the potential to be used as amodel in an assay combining functionality and binding assessment.

In the final experiments before combining the Tag-lite® assay with theluciferase assay, the optimal concentrations of the primary andsecondary antibodies were found out in a luciferase assay. First,Antibody-III as the primary antibody was titrated in five concentrationsbetween 2.5 and 40 nM, whereas the ratio from the primary to thesecondary antibody was kept constant in a 1:4 ratio. Besides the stablyand transiently transfected Receptor X expressing HEK NFκB-Luc-GFPcells, also the transiently supertransfected cells were tested (FIG. 17).

The transiently supertransfected cells show a very high bioluminescentsignal compared to the other two cell lines. Additionally, among allcell lines it can be clearly seen that a concentration of 40 nM of theprimary antibody showed the best result. Thus, this concentration forAntibody-III was kept constant in the following assay, whereas theratios to the secondary antibody have been changed from a 1:1 ratio to a1:5 ratio. Moreover, the secondary antibody which was usually used in aluciferase assay (anti-human IgG Fcγ-specific goat IgG F(ab)2 fragments)for crosslinking was compared to the d2-labeled secondary antibody(anti-human-IgG-d2) which was commonly used in a Tag-lite® experiment(FIG. 18 ). FIG. 18 shows that the bioluminescent signal was strongerwhen crosslinking with the d2-labeled anti-human-IgG. There, aconcentration of 120 nM (1:3 ratio) for the secondary antibody isalready enough. For the anti-hu IgG Fcγ-specific goat IgG F(ab)2, aconcentration of 200 nM (1:5 ratio) showed the best result.

Combination Assay with Final Conditions

All necessary parameters to combine a binding assay with a functionalityassay were evaluated and defined.

Transiently supertransfected Receptor X expressing HEK NFκB-Luc-GFPcells were used to perform the combination assay. Therefore, 10 000cells per well were mixed with Antibody-III and the secondary antibodyin a 384-well-plate. An indirect Tag-lite® binding assay was performedover a period of 4 h (FIG. 19 ). 24 h after activation of Receptor X,the cells were lyzed using the ONE-Glo™ luciferase reagent. As thisreagent also contains the luciferase substrate, the light released fromthe oxidation of luciferin to oxyluciferin was measured by theluminometer (FIG. 20 ). The KD determined in FIG. 28 was 0.10 nM±0.01 nM(R2=0.98) for the HEK NFκB-Luc-GFP-Receptor X-Tb supertransfected cells.

FIG. 20 shows that with an increasing concentration up to 10 nM of theprimary antibody, the luminescent signal was increasing, too. Thus, thecombination of the primary and secondary antibody resulted in anactivation of the NFκB pathway.

In conclusion, Antibody-III was able to bind to Receptor X and it alsoshowed a good functionality.

While there are shown and described presently preferred embodiments ofthe invention, it is to be distinctly understood that the invention isnot limited thereto but may be otherwise variously embodied andpracticed within the scope of the following claims.

Discussion

Evaluation of the Cell Lines

Prior to transfection of the SNAP-Receptor X plasmid, HEK NFκB-Luc-GFPand HeLa NFκB-Luc were tested in a luciferase assay. The signal of theHEK NFκB-Luc-GFP cells was significantly higher than it was detectedwith the HeLa NFκB-Luc cells, but those two cell lines should not becompared, since their growth rates are totally different. The sameamount of cells was seeded the day before activation. Since HEKNFκB-Luc-GFP cells divide much faster, more cells could be activated thenext day. For further experiments, HeLa NFκB-Luc cells have to be seededin a higher cell number than HEK NFκB-Luc-GFP cells. However, since bothcell lines could be activated well, they were used for transfection ofthe target receptor fused to the SNAP-tag®.

Protein-Protein Interaction Studies

In an indirect Tag-lite® binding assay, Antibody-I was compared toAntibody-II. The difference between those antibodies was an exchange ofone amino acid in the complementarity determining regions (CDRs) of thelight chain of Antibody-I. This must be the reason why Antibody-I bindsto its target unlike Antibody-II.

Instead of using Tag-lite® reaction buffer, also FluoroBrite DMEM/10%FBS and DMEM/10% FBS can be used. PBS in contrast or DMEM do not supportthe binding of the antibody to the target receptor. DMEM and DMEM/10%FBS in comparison was a good example, that FBS increases the binding.FBS as a media supplement delivers nutrients for the cells and containsgrowth factors. Probably the increase in binding when using mediumcontaining FBS was just due to a lower cell death or better cell growth.The explanation why PBS decreases the binding was probably the same andwhen incubating the cells 4 h in PBS they might start binding. Thecomposition of Tag-lite® reaction buffer is not known, but it seems thatit supported binding of two proteins and kept the cells alive.

After a transient transfection of the SNAP-Receptor X plasmid on HEKNFκB-Luc-GFP cells, the receptor stability was tested by performingTag-lite® assays every day after thawing the cells. A strong decreasefrom day 0 (directly after thawing) to day 1 was observed. But this doesnot necessarily mean that the receptors on the cell surface got lost.The cells were just transiently transfected, thus, after proliferationthe daughter cells do not have the gene for expressing Receptor X. Sincethe assay was performed by using every day the same amount of cells, itcould be that many of the cells used in the assay at day 1 are thosedaughter cells which do not have this receptor. Receptor X expressingcells at day 2 and 3 were totally overgrown by non-Receptor X expressingdaughter cells. This assumption also fits to the daily measurements ofthe terbium signal and the viability. At day 0, a high terbium signalwas determined while the viability was low. Thus, directly afterfreezing the cells were not that healthy, but most of the cellsexpressed Receptor X. From day to day the cells proliferated resultingin more healthy daughter cells but without having the labeled targetreceptor on their cell surface. Another assumption would beinternalization and degradation of the receptors.

For the combination assay of binding and functionality, this result wasnot of any concern, since the cells are activated directly afterfreezing. Then, the binding was measured over a period of 4 h and forthe measurement of the functionality after a few hours the presence ofthe receptors on the cell surface does not play any role. The period ofincubation after activating the cells and measuring functionality wasnecessary to obtain a high luciferase expression.

Luciferase Assay

Antibody-I which showed a good binding in the Tag-lite® assay was usedfor the initial luciferase assay after the transient transfection andlabeling. In addition, the NFκB pathway was also tried to be activatedby TNFα. The activation by TNFα worked quite well. This demonstratedthat the transfection and labeling of the cells did not modify the cellsand the NFκB pathway still works. There were also cells used in theassay without activation that served as a blank which was subtractedfrom the signal. This confirmed that the cells do not express anyluciferase constitutively. Only a weak luminescent signal was observedwhen activating the cells with Antibody-I and the secondary antibody.This was a good example to show the necessity to perform a combinationassay, since Antibody-I was determined to be one of the best binders.

In another Luciferase assay, two different incubation times (6 h and 48h) were compared using stably and transiently transfected Receptor Xexpressing HEK NFκB-Luc-GFP cells. It was clearly seen that moreluciferase was expressed in the stably transfected cells after 48 hwhich resulted in a stronger luminescent signal. Unfortunately, thetransiently transfected cells showed only a very weak luminescentsignal. The transiently transfected cells express Receptor X only fusedto a SNAP-tag®. Since the activation of the NFκB pathway occurs onlyupon oligomerization of the ReceptorX, the size of the SNAP-tag® (20kDa) might lead to steric hindrance and inhibition of theoligomerisation.

Indirect WGA-HTRF

WGA enables to perform cell based protein-protein interactionmeasurements in a new way by avoiding the direct labeling of receptorsexpressed on the cell surface. An approach to avoid the SNAP-tag® and toallow the use of stably transfected cells for the combination assay wasto label them with terbium by using WGA. Thus, an indirect HTRF assaycould be performed. The KD value was with 0.15 nM±0.03 nM in the samerange than it was determined in the normal Tag-lite® experiments ontransiently transfected Receptor X expressing HEK NFκB-Luc-GFP cells.Thus, WGA as a labeling tool would also be useful in this set-up toperform a combination assay but could not be further evaluated due totime limitations.

Evaluation of a Cell Line Suitable for Combining Functionality andBinding Assessment

A transient supertransfection of stably transfected HEKNFκB-Luc-GFP-Receptor X cells was the second approach, which enables toperform a normal indirect Tag-lite® binding experiment. Besides testingthem in a binding assay, also the luciferase assay was evaluated withthis cell line. Therefore, those cells were used to find out the optimalconcentration of the primary antibody and simultaneously they werecompared to transiently and stably transfected Receptor X expressing HEKNFκB-Luc-GFP cells. For every concentration of Antibody-III, thesupertransfected cells show clearly the strongest luminescent signal.One reason why the supertransfected cells show a higher signal comparedto stable cells is that there were more target receptors present on thecell surface. Thus, more antibodies could bind and more crosslinkingcould take place, resulting in a stronger activation of the NFκBpathway. The signal of the stably transfected cells was in general inthis assay extremely low. The reason was probably a wrong handling inpassaging cells. If they are splitted very late and the cells have ahigh confluency, it was observed that the cells cannot be activated thatwell. Either some cells lost the receptors on their cell surface or theydownregulated signaling events somehow.

It is also assumed that the signal of the transiently supertransfectedcells was higher than of the transiently transfected cells due to sterichindrance. The supertransfected cells overexpress Receptor X on theircell surface. Some of them are fused to a SNAP-tag®, since they weretransiently transfected with a SNAP-Receptor X plasmid, but some of themdo not have the SNAP-tag®, since the stably transfected Receptor Xexpressing HEK NFκB-Luc-GFP cell line was used for the transienttransfection of SNAP-Receptor X. Probably, the activation of this cellswas better because of the reason that there was less steric hindrancewhen oligomerisation of receptors carrying the SNAP-tag® with receptorswithout SNAP-tag® took place.

In addition, two secondary antibodies were compared on transientlysupertransfected Receptor X expressing HEK NFκB-Luc-GFP cells. The onewhich was usually used in a luciferase assay (anti-human IgGFcγ-specific goat IgG F(ab)2 fragments) showed a lower signal comparedto the d2-labeled secondary antibody (anti-human-IgG-d2) which wascommonly used in a Tag-lite® experiment. A possible reason is that theanti-human-IgG-d2 is a polyclonal antibody. Polyclonal antibodies areideally suited as a secondary antibody, since those antibodies are notrestricted to one epitope on their target and can therefore crosslinkvarious constructs at the same time. The formerly used secondaryantibody is a monoclonal (Fab)2 fragment binding only to one specificepitope which is not ideal for achieving a good hypercrosslinking.

Finally, the transiently supertransfected Receptor X expressing HEKNFκB-Luc-GFP cells were used as a model for the combination assay.

Conclusion

It was possible to align the parameters of both individual assays,luciferase and Tag-lite® assay, to develop a new assay, combining bothin one well. For instance, the Tag-lite® assay can be performed at 37°C. instead of incubation at room temperature. Additionally, medium couldbe used instead of Tag-lite® reaction buffer and also the amount ofcells that are seeded per well can vary in a large extent withoutchanging the results. Moreover, down-scaling of the ONE-Glo™ Luciferaseassay system worked well. Thus, the combination assay could be performedin a 384-well-plate, which was also used for the Tag-lite® experimentsin the past. Advantages of this scale are not only the lower amounts ofreagents that are needed, resulting in a drop in the overall costs, butalso its suitability for performing HTS.

The final combination assay was performed with transientlysupertransfected cells, since there was no stable cell line availablecontaining the SNAP tag additionally to the target receptor. For futureexperiments using a combination assay, it would be possible to generatecell lines with that tag or to supertransfect them, as well. Inaddition, cells without the SNAP-tag® could be used by performing aWGA-HTRF.

In conclusion, the development of the combination assay is not onlyuseful for the NFκB signaling pathway, there is also a potential toextend the assay to other functionality readouts, e.g. cytokine releaseor additional signaling pathways.

Example 6: AlphaScreen

For the evaluation of the different assays huDR5 specific antibodyDrozitumab was used. HuDR5 was expressed by HEK EBNA cells. For theAlphaScreen based method, biotinylated WGA was bound to Streptavidindonor beads (PerkinElmer) and binding of Drozitumab to DR5 was detectedwith ProtA acceptor beads (PerkinElmer) which bind to the Fc portion ofantibodies.

It is noteworthy that all pipetting steps and measurements were done ina dark room because the beads are light sensitive.

Defining the Assay Window of the AlphaScreen

To define the assay window an AlphaScreen assay was done with 1.4 nMWGA-biotin and 10000 cells. PerkinElmer recommended a concentration of10 μg/ml of each bead, acceptor and donor. First, 10 μg/ml ofStreptavidin donor beads were mixed with 1.4 nM of WGA-biotin andincubated for 30 min. Meanwhile, the dilution row of Drozitumab wasprepared starting with a concentration of 1200 nM to 0.0011 nM in 1 in 4dilution steps. The HEK EBNA cells expressing huDR5 SNAP Tag werethawed, washed with 10 ml of 1×PBS and centrifuged for 8 min and 350 g.The supernatant was discarded. The pellet was resuspended in anappropriate volume to get 1 mio cells/ml. The Streptavidin beads labeledwith WGA-biotin were added to the resuspended cells and incubated for 30min at room temperature. While incubating the cells, 5 μl of the dilutedantibodies were transferred to a 384-well plate in duplicates and 5 μlof the ProteinA acceptor beads (10 μg/ml) were added. The 384-plate wasincubated for 30 min. The cells were filled up to 10 ml with 1× reactionbuffer and centrifuged for 8 min at 350 g. The supernatant was discardedand the pellet was resuspended in an appropriate volume with 1× reactionbuffer to get 1.0 mio cells/ml. 10 μl of the WGA-streptavidin beadslabeled cells were transferred to each well of a 384-well plate. Theblank was prepared with 10 μl WGA-streptavidin labeled cells, 5 μl ofProteinA acceptor beads and 5 μl of 1× reaction buffer. All measurementswere done in duplicates. The 384-well plate was measured after 1 h, 2 h,3 h and 4 h with the Tecan M1000 Pro reader using the AlphaScreentemplate. The raw data was evaluated in Excel by subtracting the blankand further analyzing it in GraphPad Prism.

Evaluation of Cell Number and WGA-Biotin Per Well

First, 10 μg/ml of Streptavidin donor beads were mixed with either 1.4nM or 2.8 nM of WGA-biotin and incubated for 30 min. Meanwhile, thedilution row of Drozitumab was prepared starting with a concentration of6.25 nM to 0.020 nM in 1 in 2 dilution steps. The HEK EBNA cellsexpressing huDR5 SNAP Tag were thawed, split in two 15 ml Falcon tubesand washed with 10 ml of 1×PBS and centrifuged for 8 min and 350 g. Thesupernatant was discarded. The pellet of the two vials was resuspendedin an appropriate volume to get 1 mio cells/ml or 2.0 mio cells/ml. TheStreptavidin beads labeled with WGA-biotin were transferred to theresuspended cells and incubated for 30 min at room temperature. Whileincubating the cells, 5 μl of the diluted antibodies were transferred toa 384-well plate in duplicates and 5 μl of the ProteinA acceptor beads(10 μg/ml) were added. The 384-plate was incubated for 30 min. The cellswere filled up to 10 ml with 1× reaction buffer and centrifuged for 8min at 350 g. The supernatant was discarded and the pellet wasresuspended in an appropriate volume with 1× reaction buffer to get 1.0mio cells/ml and 2.0 mio cells/ml. 10 μl of the WGA-streptavidin beadslabeled cells were transferred to the 384-well plate. The blank wasprepared with 10 μl WGA-streptavidin labeled cells, 5 μl of ProteinAacceptor beads and 5 μl of 1× reaction buffer. All measurements weredone in duplicates. The 384-well plate was measured after 1 h, 2 h, 3 hand 4 h with the Tecan M1000 Pro reader using the AlphaScreen template.The raw data was evaluated in Excel by subtracting the blank and furtheranalyses in GraphPad Prism.

Evaluation of Different Acceptor/Donor Pairs

To define which acceptor/donor pair works best, an AlphaScreen assay wascarried out with 2.8 nM WGA-biotin and 20000 cells. First, 10 μg/ml ofStreptavidin donor beads or 10 μg/ml of Streptavidin acceptor beads weremixed with 2.8 nM of WGA-biotin and incubated for 30 min. Meanwhile, thedilution row of Drozitumab and AbY was prepared starting with aconcentration of 25 nM to 0.024 nM in 1 in 2 dilutions. The HEK EBNAcells expressing huDR5 SNAP Tag were thawed and split in two 15 mlFalcon tubes, one for each Streptavidin bead (donor or acceptor). Thecells were washed with 10 ml of 1×PBS and centrifuged for 8 min and 350g. The supernatant was discarded. The pellet of the two vials wasresuspended in an appropriate volume to get 2.0 mio cells/ml. TheStreptavidin beads labeled with WGA-biotin were transferred to theresuspended cells and incubated for 30 min at room temperature. Whileincubating the cells, 5 μl of the diluted antibodies were transferred toa 384-well plate in duplicates and either 5 μl of the ProteinA donorbeads (10 μg/ml) or ProteinA acceptor beads (10 μg/ml) was added. The384-plate was incubated for 30 min. The cells were filled up to 10 mlwith 1× reaction buffer and centrifuged for 8 min at 350 g. Thesupernatant was discarded and the pellet of both vials was resuspendedin an appropriate volume with 1× reaction buffer to get 2.0 miocells/ml. 10 μl of the WGA-streptavidin beads labeled cells weretransferred to the 384-well plate. The blank was prepared with 10 μlWGA-streptavidin labeled cells, 5 μl of ProteinA beads and 5 μl of 1×reaction buffer. All measurements were done in duplicates. The 384-wellplate was measured after 1 h, 2 h, 3 h and 4 h with the Tecan M1000 Proreader using the AlphaScreen template. The raw data was evaluated inExcel by subtracting the blank and analyzed in GraphPad Prism.

AlphaScreen with Final Conditions

First, 10 μg/ml of Streptavidin acceptor beads were mixed with 2.8 nM ofWGA-biotin and incubated for 30 min. Meanwhile, the dilution row ofDrozitumab was prepared starting with a concentration of 2.5 nM to 0.020nM. The dilution was done in 1 in 2 dilution steps. The HEK EBNA cellsexpressing huDR5 SNAP Tag were thawed, washed with 10 ml of 1×PBS andcentrifuged for 8 min and 350 g. The supernatant was discarded. Thepellet was resuspended in an appropriate volume to get 2.0 mio cells/ml.The Streptavidin beads labeled with WGA-biotin were transferred to theresuspended cells and incubated for 30 min at room temperature. Whileincubating the cells, 5 μl of the diluted antibodies were transferred toa 384-well plate in duplicates and 5 μl of the ProteinA donor beads (10μg/ml) was added. The 384-plate was incubated for 30 min. The cells werefilled up to 10 ml with 1× reaction buffer and centrifuged for 8 min at350 g. The supernatant was discarded and the pellet was resuspended inan appropriate volume with 1× reaction buffer to get 2.0 mio cells/ml.10 μl of the WGA-streptavidin beads labeled cells were transferred tothe 384-well plate. The blank was prepared with 10 μl WGA-streptavidinlabeled cells, 5 μl of ProteinA donor beads and 5 μl of 1× reactionbuffer. All measurements were done in triplicates. The 384-well platewas measured after 1 h, 2 h, 3 h and 4 h with the Tecan M1000 Pro readerusing the AlphaScreen template. The raw data was evaluated in Excel bysubtracting the blank. K_(D) value was calculated by nonlinearregression.

Evaluation of the AlphaScreen Raw Data

The data was normalized to the background. The normalized data wereevaluated by nonlinear regression to determine the binding curve and theK_(D) value by the software GraphPad Prism 6.0. The binding curve wasfitted using a sigmoidal dose-response (variable slope) according to thefollowing equation:

$Y = {{Bottom} + {\frac{\left( {{Top} - {Bottom}} \right)}{1 + 10^{{({{\log\;{EC50}} - X})}*{Hill}\mspace{14mu}{Slope}}}.}}$

The K_(D) was determined using a one site binding model according to thefollowing equation:

$Y = {\frac{B_{\max}*X}{\left( {K_{D} + X} \right)}.}$ResultsDefining the Assay Window of the AlphaScreen

In the first assay the number of huDR5 expressing HEK EBNA cells perwell, the amount of biotinylated WGA per well and the incubation time ofWGA-biotin with the cells was adopted from the WGA-HTRF. 10000 cells and1.4 nM WGA-biotin were used per well. Both antibodies were dilutedstarting from 300 nM to 3.0*10⁴ nM final in well in 1 in 4 dilutions.

The first test of the AlphaScreen assay showed that the idea of theassay setup should work. However, the curve of Drozitumab showed arelatively small assay window starting with a concentration of 0.018 nMto 19 nM.

Evaluation of Cell Number and WGA-Biotin Per Well

To improve the assay in a next step the cell number was set to 10000 and20000 cells per well and the amount of WGA-biotin was set to either 1.4nM or 2.8 nM per well. To get a better assay window the dilution stepswere decreased to 1 in 2 rather than 1 in 5 starting with aconcentration of 50 nM final in the well. Both antibodies were dilutedstarting from 50 nM to 0.049 nM final in well in 1 in 2 dilutions.

Both assay setups using 1.4 nM WGA-biotin per well showed only a slightor no binding signal. The one with 10000 cells per well and 2.8 nMWGA-biotin per well showed a weak binding curve. The best result wereobtained with 20000 cells and 2.8 nM WGA-biotin per well.

Evaluation of Different Acceptor/Donor Pairs

To perform an AlphaScreen assay it is possible either to useStreptavidin donor beads with ProtA acceptor beads or Streptavidinacceptor beads with ProtA donor beads. To find out the bestdonor/acceptor pair they were compared to each other Both antibodieswere diluted starting from 1.6 nM to 0.006 nM final in well in 1 in 2dilutions. The two different donor/acceptor pairs showed nearly the samebinding curve. Even the difference in their R² value was almost thesame. The assay measured with Streptavidin donor beads and ProtAacceptor beads had a R² value of 0.99 and the assay measured withStreptavidin acceptor beads paired with ProtA acceptor beads had a R²value of 0.99.

AlphaScreen with Final Conditions

The result of the evaluation of the AlphaScreen was to run theAlphaScreen with the following set up (table 2).

TABLE 2 Finally evaluated parameters to perform a AlphaScreen assay.Parameter Conditions WGA-biotin per well 2.8 nM Cell count per well20000 cells Amount of streptavidin donor beads 10 μg/ml Amount of ProtAacceptor beads 10 μg/ml Incubation time of streptavidin acceptor 30 minbeads and WGA-biotin Incubation time of acceptor beads labeled 30 minWGA and cells Incubation time of antibody dilutions with 30 min ProtAacceptor beads

With the final condition evaluated for the AlphaScreen assay the K_(D)value for the binding of Drozitumab to huDR5 was determined (FIG. 21 ).

The sigmoidal fitting of Drozitumab showed a sigmoidal shaped bindingcurve The K_(D) determined by the nonlinear regression was 0.084nM+/−0.0053 nM. Since the AbY showed no binding to huDR5 there was noK_(D) value calculated.

Like in the WGA-HTRF assay the result of the assay evaluation has to beconfirmed using another antibody and antigen and the K_(D) of AbZ to itstarget receptor Z was determined (FIG. 22 ).

As in the previous assays the negative control antibody AbY showed nobinding. The K_(D) value was determined by nonlinear regression. TheK_(D) of AbZ—receptor Z binding was 0.26 nM+/−0.068 nM with a R² valueof 0.96. Since the AbY is a non-binder to receptor Z there was no K_(D)value determined.

Example 7: Measuring CD3 Binding and Functionality in One Well Using aJurkat NFAT Reporter Cell Line

The Jurkat NFAT-luciferase reporter cell line is designed to monitor Tcell activation that results in modulation of the nuclear factor ofactivator T cells (NFAT) activities.

The transcription factor NFAT has important roles in T cell developmentand function. In non-activated T cells, NFAT is mainly located in thecytoplasm, in a highly phosphorylated form. After antigenic T cellstimulation via TCR/CD3 receptor complex, the Ca²⁺-dependent phosphatasecalcineurin dephosphorylates multiple phosphoserines on NFAT, leading toits nuclear translocation and downstream gene expression of T cellactivation genes.

The NFAT reporter cell was generated by clonal selection of Jurkat cellsstably transfected using a NFAT-luciferase reporter vector, whichcontains multiple repeats of a NFAT response element and a minimalpromoter upstream of the firefly luciferase coding region. Theactivation of NFAT has been confirmed following the induction of thereporter cell with PMA/ionomycin.

Methods

Labeling of Jurkat NFAT Reporter Cells with WGA-Tb:

Jurkat NFAT cells were spun down and resuspended in either PBS/1% FCS orgrowth medium to obtain 4 Mio cells/ml. Subsequently, 1 Mio cells werelabeled by adding 0.5 ng/ul WGA-Terbium and incubated for 30 min at RT.Following the incubation, the cells were washed twice with PBS bycentrifugation for 8 min at 280 g. Finally cells were resuspended ineither PBS/1% FCS or growth medium to obtain a final number of 4 Miocells/ml.

Binding:

A d2-labeled antibody binding to CD3 as well as a d2-labeled non-bindingcontrol IgG were resuspended in either PBS/1% FCS or growth medium and aserial dilution generated ranging from final 50-0.1 nM in 1:2 dilutionsteps. 40000 (10 ul) labeled cells were mixed in a 384 well with 5 ullabeled antibody and 5 ul PBS/1% FCS or growth medium. The binding assaywas incubated for 5 h at 37° C. The TR-FRET signal was measured using aM1000 Pro Reader by determining the absorbance at 665 nm as well as 615nm after a delay time of 60 sec. 40000 WGA-Tb labeled Jurkat-NFAT cellsserved as blank. The ratio of 665 nm/615 nm was determined for each wellto normalize the specific FRET signal to the donor signal in each well.Additionally, the ratio of the blank was subtracted from the ratio ofthe samples before determining the KD and EC50 values in GraphPad Prism6.0. The binding curves were fitted with nonlinear regression. Bmax andKD were determined using the “One site specific binding” model using theequation Y=“(“B”_“max” “*X”)/(“(“K”_“D” “+X)”).

Evaluation

Evaluation of the HTRF Raw Data For the analysis of the assays, the rawdata were first edited by Microsoft Excel. In a HTRF assay variations inthe results can occur from well to well due to the pipetting steps ofthe cells, medium additives and from the number of lysed cells per well.To minimize those variations, the emission of the acceptor wasnormalized to the emission of the donor signal in each well bycalculating the ratio of 665 nm to 620 nm: “ratio=” “665 nm”/“620 nm”“*10 000”. The calculated ratio values were evaluated by the softwarecalled GraphPad Prism 6.0. The binding curves were fitted with nonlinearregression. Bmax and KD were determined using the “One site—specificbinding” model using the equation: “Y=”(“B”_“max” “*X”)/(“(” “K”_“D”“+X)”).

Functional Assay—Luciferase Assay

CD3 signalling was measured from the same wells as the binding assay bylysing the cells after 6 h incubation at 37° C. Therefore, 5 μl assaymix was removed per well followed by adding 15μ One-Glo Luciferasereagent. After incubation for 5-10 min at RT in the dark, theluminescence signal was measured with the M1000Pro Reader with a 1000 msintegration time. As a blank, the signal of the lysed non-activatedcells with the luciferase reagent was subtracted. The measurement wasdone in triplicates.

Results

Binding Assay:

CD3 binding could be detected in growth medium (FIG. 23 a ) as well asPBS/1% FCS (FIG. 23 b ). The non-binding control does not show anybinding in this assay system. The KD value for the binding of anti-CD3to CD3 in growth medium is approximately 7 fold higher than in PBS/1%with 7 nM compared to 0.98 nM respectively.

Functional Assay:

The results show that the signaling upon CD3 binding did only work ingrowth medium (FIG. 24 a ) and not in PBS/1% FCS (FIG. 24 b ) althoughsimilar binding was observed under both conditions. The EC50 of theluciferase activity was 1.4 nM.

CONCLUSION

The CD3 binding and functionality assessment experiment in the JurkatNFAT reporter cell line is an additional example of an assay systemwhere binding and functionality could be measured in a high-throughputformat in one well.

The invention claimed is:
 1. An in vitro assay method for determiningthe binding and functionality of a test antibody specifically binding toa target antigen comprising the following steps i) providing cells whicha) expresses the target antigen on their surface, b) are covalently ornoncovalently labelled with an energy donor compound, and c) comprise areporter gene under the control of a response element of the targetantigen; ii) adding the test antibody; iii) measuring the binding to thetarget antigen by determining the energy transfer, wherein an energyacceptor compound is covalently or noncovalently conjugated either tothe test antibody or to a secondary antibody binding to the testantibody; and iv) determining functionality of the test antibodycorrelating the level of the expression of the reporter gene with thelevel of target antigen activation or inhibition, wherein the binding tothe target antigen and the functionality of the test antibody ismeasured in the same vial, and wherein the functionality is a biologicalactivity of the test antibody.
 2. The method of claim 1, wherein theenergy donor and acceptor compound are a fluorescent resonance energytransfer (FRET) energy donor and acceptor compound and the energytransfer determined in step iii) is fluorescent resonance energytransfer (FRET).
 3. The method of claim 2, wherein the FRET is timeresolved FRET.
 4. The method of claim 1, wherein the energy donor andacceptor compound are a bioluminescence energy transfer (BRET) energydonor and acceptor compound and the energy transfer determined in stepiii) is bioluminescence energy transfer (BRET).
 5. The method of claim1, wherein the energy donor and acceptor compound are an AmplifiedLuminescent Proximity Homogeneous Assay acceptor and donor bead and theenergy transfer determined in step iii) is an energy transfer from asinglet oxygen to an thioxene derivative within the acceptor bead. 6.The method of claim 1, wherein the target antigen is covalently ornoncovalently labelled with the energy donor compound.
 7. The method ofclaim 1, wherein the energy donor compound is covalently ornoncovalently linked to wheat germ agglutinin (WGA).
 8. The method ofclaim 1, wherein the reporter gene is selected from a gene coding for afluorescent protein or a gene coding for an enzyme whose catalyticactivity can be detected.
 9. The method of claim 8, wherein the reportergene is coding for green fluorescent protein (GFP) or luciferase. 10.The method of claim 1, wherein the target antigen is a cell surfacereceptor.
 11. The method of claim 1, wherein steps iii) and iv) areperformed consecutively or simultaneously.
 12. The method of claim 1,wherein the target antigen and the response element are part of theNF-κB pathway.
 13. The method of claim 12, wherein the response elementcomprises at least one DNA repeat with a DNA sequence of SEQ ID NO: 1,2, 3, 4 or
 5. 14. The method of claim 12, wherein the response elementcomprises a DNA sequence of SEQ ID NO 6, 7, 8 or
 9. 15. The method ofclaim 1, wherein the cells are transfected with an expression vectorcomprising a DNA sequence coding for the reporter gene under the controlof the target antigen response element.
 16. The method of claim 1,wherein the target antigen is fused to a SNAP tag.
 17. The method ofclaim 16, wherein the target antigen is introduced into the cells bytransfection of the cells with a vector that encodes the target antigen.18. The method of claim 1, wherein the energy donor compound is terbium.19. The method of claim 1, wherein the cells are grown in the presenceof a growth medium or a growth media supplement.
 20. The method of claim1, wherein the test antibody is added at a concentration of at least 40nM.
 21. The method of claim 1, wherein the ratio of the test antibody tothe secondary antibody is at least 1:3.
 22. The method of claim 1,wherein the cells are mammalian cells.
 23. The method of claim 17,wherein the cells are stably transfected and/or transiently transfectedwith the vector encoding the target antigen.
 24. The method of claim 17,wherein the cells are transfected by lipofection.
 25. The method ofclaim 1, wherein the binding to the target antigen and the functionalityof the test antibody are measured after incubation of the cell line withthe test antibody for at least 4 hours at 37° C.
 26. The method of claim1, wherein determining functionality of the test antibody comprisesreducing the volume of the cells before adding a detection reagent tothe cells.